Novel human leucine-rich repeat domain containing protein HLLRCR-1

ABSTRACT

The present invention provides novel polynucleotides encoding HLLRCR-1 polypeptides, fragments and homologues thereof Also provided are vectors, host cells, antibodies, and recombinant and synthetic methods for producing said polypeptides. The invention further relates to diagnostic and therapeutic methods for applying these novel HLLRCR-1 polypeptides to the diagnosis, treatment, and/or prevention of various diseases and/or disorders related to these polypeptides, particularly nervous system diseases and/or disorders. The invention further relates to screening methods for identifying agonists and antagonists of the polynucleotides and polypeptides of the present invention.

This application claims benefit to provisional application U.S. Ser. No.60/328,478 filed Oct. 11, 2001. The entire teachings of the referencedapplication are incorporated herein by reference.

FIELD OF THE INVENTION

The present invention provides novel polynucleotides encoding HLLRCR-1polypeptides, fragments and homologues thereof. Also provided arevectors, host cells, antibodies, and recombinant and synthetic methodsfor producing said polypeptides. The invention further relates todiagnostic and therapeutic methods for applying these novel HLLRCR-1polypeptides to the diagnosis, treatment, and/or prevention of variousdiseases and/or disorders related to these polypeptides, particularlynervous system diseases and/or disorders. The invention further relatesto screening methods for identifying agonists and antagonists of thepolynucleotides and polypeptides of the present invention.

BACKGROUND OF THE INVENTION

Recently, a class of cell surface proteins have been described in bothplants and animals that are involved in pathogen perception, MHC classII trans-activation, inflammation and the regulation of apoptosis(Inohara, N., Nunez, G, Cell, Death, Differ., 6(9):823-4, (1999);Inohara, N., Koseki, T., del, Peso, L., Hu, Y., Yee, C., Chen, S.,Carrio, R., Merino, J., Liu, D., Ni, J., Nunez, G, J. Biol, Chem. 21.,274(21):14560-7, (1999); Inohara, N., Nunez, G, Cell, Death, Differ.,7(5):509-10, (2000); Harton, J A., Ting, J P, Mol, Cell, Biol.,20(17):6185-94, (2000); Dixon, J., Brakebusch, C., Fassler, R., Dixon, MJ. Hum, Mol, Genet. 12., 9(10):1473-80, (2000)). All of these proteinsare modular in nature containing one or several domains that function incaspase recruitment (CARD), nucleotide binding and protein-proteininteractions. Proteins within this group have also been found to play arole in cell adhesion during various developmental processes.

A common theme in all of these proteins are the presence of aleucine-repeat repeat (LLR) in the carboxy terminus of the polypeptidechain. LLRs are short protein modules characterized by a periodicdistribution of hydrophobic amino acids, especially leucine residuesseparated by hydrophilic residues [Sean, 1996]. The basic structure ofthe repeat is as follows:X-L-X-X-L-X-L-X-X-N-X-a-X-X-X-a-X-X-L-Xwhere X is any amino acid, L is leucine, N is asparagine and “a” denotesan aliphatic residue. The asparagine at position 10 can be replaced bycysteine, threonine or glutamine. The average repeat length is 24 aminoacids but it can vary between 22 to 29 amino acids, though some LRRmotifs have been reported to be at short as 20 amino acids. The motifoften consists of leucine or other aliphatic residues at positions 2, 5,7, 12, 16, 21, and 24 and asparagine, cysteine or threonine at position10. X-ray structure determination of LRR motifs suggests that each LRRis composed of a beta-sheet and an alpha-helix. The largest subfamily ofproteins that contain a leucine-rich domain are extracellular proteinshaving the following motif: LxxLxxLxLxxNxLxxLPxxOFxx, where “x” is anyamino acid and “O” is a non-polar residue (Kajava, J. Mol. Biol. 277:519 (1998)).

In transmembrane proteins, LLRs and their flanking sequence always occurin the presumed extracellular portions. In these situations the LLRs aregenerally flanked on either side by cysteine-rich regions. In general,these cysteines are present in the oxidized disulphide link form. Anexample of a transmembrane protein containing a LRR is Toll, aDrosophila gene the functions in establishment of dorsal-ventralpatterning. Dominant, ventralizing mutants have been described that mapto the cysteine-rich regions surrounding the LLR domain [Schneider,1991]. Thus, the cysteine regions associated with LLRs act to regulatereceptor activity. The LLRs themselves within the Toll protein have beenshown to function in heterotypic cell adhesion, a process required forproper motoneuron and muscle development [Halfon, 1995]

Another Drosophila LLR containing transmembrane protein, 18 wheeler,which is regulated by homeotic genes also promotes heterophilic celladhesion in cell migration events during development (Eldon, E., Kooyer,S., D'Evelyn, D., Duman, M., Lawinger, P., Botas, J., Bellen, H,Development., 120(4):885-99, (1994)). Mammalian CD14, which bindslipopolysaccharide (LPS), and signals through NF-κB, is thought to haveanalogies to the Toll signal transduction pathway. CD14 also contains aregion of LLRs that have been shown in deletion mutants to beresponsible for LPS binding.

Slit is another LLR containing Drosophila secreted protein thatfunctions in the development of the midline glial cells and thecommissural axon tracts the cross the midline. This is presumablyaccomplished by cell adhesion events (Jacobs, J R, J. Neurobiol.,24(5):611-26, (1993)). Mammalian homologues of Drosophila slit have beenshown to bind the heparan sulfate proteoglycan, glypican-1 (Liang, Y.,Annan, R S., Carr, S A., Popp, S., Mevissen, M., olhs, R K., olis, R U,J. Biol, Chem. 18., 274(25): 17885-92, (1999)). In general, heparansulfate proteoglycans have been shown to accummulate in Alzheimer'sdisease brains and specifically, glypican-1 is component of both senileplaques and neurofibrillary tangles (Verbeek, M M., Otte, Holler, I.,van, den, Born, J., van, den, Heuvel, L P., David, G., Wesseling, P.,de, Waal, R M, Am. J. Pathol., 155(6):2115-25, (1999)). Heparan sulfateproteoglycans are also implicated in the regulation of cytokinesignaling in B cells through the activation of CD40 (van, der, Voort,R., Taher, T E., Derksen, P W., Spaargaren, M.,van, der, Neut, R., Pals,S T, Adv, Cancer, Res., 79:39-90, (2000)).

p37NB is a 37 kDa LRR protein identified in human neuroblastoma cells(Kim, D. et al. (1996) Biochim. Biophys. Acta 1309: 183-188). Northernblot hybridization and RT-PCR studies show that p37NB is differentiallyexpressed in several neuroblastoma cell lines. A related LRR protein,PRELP, is characterized as a 42 kDa secreted protein (Bengtsson, E. etal. (1995) J. Biol. Chem. 270: 25639-25644). PRELP consists of 10 LRRmotifs ranging in length from 20 to 26 residues with asparigine atposition 10. Northern analysis shows differential expression of PRELP invarious tissues.

In addition, leucine-rich repeat containing proteins have also beenimplicated in various aspects of protein-protein interaction, such ascell-to-cell commnunication and signal transduction (for a review, seeKobe and Deisenhofer, TIBS 19: 415 (1994); Kobe and Deisenhofer, Curr.Opin. Struct. Biol. 5: 409 (1995); Kajava, J. Mol. Biol. 277: 519(1998)). Proteins that contain an LRR motif include hormone receptors,enzyme subunits, cell adhesion proteins, and ribosome-binding proteins.

A subfamily of the LRR superfamily, referred to as the Small LeucineRich Proteoglycan family, illustrates the critical functions fulfilledby proteins containing an LRR motif. Members of this subfamily arebelieved to play essential biological roles during inflammation andcancer invasion, a regulatory role in collagen fibrinl formation,suppression of the malignant phenotype of cancer cells, and aninhibition of the growth of certain normal cells (see, for example,Iozzo, Annu. Rev Biochem. 67: 609 (1998)).

Kajava, et al., J. Mol. Biol. 277: 519 (1998), divided the LRRsuperfamily into subfamilies characterized by different lengths andconsensus sequences of the leucine-rich repeats. Based upon thisstructural analysis, Kajava concluded that LRR proteins of differentsubfamilies probably emerged independently during evolution, indicatingthat proteins with the LRR motif provide a unique solution for a widerange of biological functions.

LLR containing proteins have been identified in prokaryotes, plants,yeast and mammals. Although such proteins were initially thought to besecreted proteins, it is now appreciated that they inhibit a variety ofcellular locations and participate in a diverse set of criticalfunctions in development and cellular homeostasis.

Such LRRs, being extracellular, are capable of directing protein-proteininteractions with other receptors involved in apoptosis, inflammationand immune responses. LLR containing proteins may also bind otherextracellular ligands derived from infectious agents and participate inthe triggering and or modulating immune responses.

The recently cloned Nogo receptor (Fournier et al., 2001), is a 473amino acid protein (Genbank Accession number: AAG53612). This receptoris brain specific, is associated with the cell surface via aGPI-linkage, and possesses eight leucine rich repeat domains and acysteine rich LRR carboxy-terminal flanking domain. Expression of thisreceptor confers responsiveness to Nogo-66, which binds to Nogoreceptor, in unresponsive cells. Identification of small molecules thatblock the inhibitory effects of Nogo on axonal regeneration can be usedfor therapeutic treatment of neurodegenerative conditions.

Using the above examples, it is clear the availability of a novel clonedleucine-rich repeat containing protein provides an opportunity foradjunct or replacement therapy, and are useful for the identification ofleucine-rich repeat containing protein agonists, or stimulators (whichmight stimulate and/or bias leucine-rich repeat containing proteinaction), as well as, in the identification of leucine-rich repeatcontaining protein inhibitors. Hence it can be reasoned that agonistsand antagonists for these LLR containing proteins will be useful fortherapeutic purposes

The present invention also relates to recombinant vectors, which includethe isolated nucleic acid molecules of the present invention, and tohost cells containing the recombinant vectors, as well as to methods ofmaking such vectors and host cells, in addition to their use in theproduction of HLLRCR-1 polypeptides or peptides using recombinanttechniques. Synthetic methods for producing the polypeptides andpolynucleotides of the present invention are provided. Also provided arediagnostic methods for detecting diseases, disorders, and/or conditionsrelated to the HLLRCR-1 polypeptides and polynucleotides, andtherapeutic methods for treating such diseases, disorders, and/orconditions. The invention further relates to screening methods foridentifying binding partners of the polypeptides.

The present invention also relates to recombinant vectors, which includethe isolated nucleic acid molecules of the present invention, and tohost cells containing the recombinant vectors, as well as to methods ofmaking such vectors and host cells, in addition to their use in theproduction of HLLRCR-1 polypeptides or peptides using recombinanttechniques. Synthetic methods for producing the polypeptides andpolynucleotides of the present invention are provided. Also provided arediagnostic methods for detecting diseases, disorders, and/or conditionsrelated to the HLLRCR-1 polypeptides and polynucleotides, andtherapeutic methods for treating such diseases, disorders, and/orconditions. The invention further relates to screening methods foridentifying binding partners of the polypeptides.

BRIEF SUMMARY OF THE INVENTION

The present invention provides isolated nucleic acid molecules, thatcomprise, or alternatively consist of, a polynucleotide encoding thefull-length HLLRCR-1 protein having the amino acid sequence shown inFIGS. 1A-C (SEQ ID NO:2) or the amino acid sequence encoded by the cDNAclone, HLLRCR-1 (also referred to as GPCR111, GPCR111-80b, and/orHLLRCAR-1), deposited as ATCC Deposit Number PTA-3434 on Jun. 7, 2001.

The present invention provides isolated nucleic acid molecules, thatcomprise, or alternatively consist of, a polynucleotide encoding analternative full-length HLLRCR-1 protein having the amino acid sequencecorresponding to amino acids 1 to about amino acid 517 of FIGS. 1A-C(SEQ ID NO:3) or the amino acid sequence encoded by the cDNA clone,HLLRCR-1, deposited as ATCC Deposit Number PTA-3434 on Jun. 7, 2001.

The present invention also relates to recombinant vectors, which includethe isolated nucleic acid molecules of the present invention, and tohost cells containing the recombinant vectors, as well as to methods ofmaking such vectors and host cells, in addition to their use in theproduction of HLLRCR-1 polypeptides or peptides using recombinanttechniques. Synthetic methods for producing the polypeptides andpolynucleotides of the present invention are provided. Also provided arediagnostic methods for detecting diseases, disorders, and/or conditionsrelated to the HLLRCR-1 polypeptides and polynucleotides, andtherapeutic methods for treating such diseases, disorders, and/orconditions. The invention further relates to screening methods foridentifying binding partners of the polypeptides.

The invention further provides an isolated full-length HLLRCR-1polypeptide having an amino acid sequence encoded by a polynucleotidedescribed herein.

The invention further relates to a polynucleotide encoding a polypeptidefragment of SEQ ID NO:2, or a polypeptide fragment encoded by the cDNAsequence included in the deposited clone, which is hybridizable to SEQID NO:1.

The invention further relates to a polynucleotide encoding a polypeptidedomain of SEQ ID NO:2 or a polypeptide domain encoded by the cDNAsequence included in the deposited clone, which is hybridizable to SEQID NO:1.

The invention further relates to a polynucleotide encoding a polypeptideepitope of SEQ ID NO:2 or a polypeptide epitope encoded by the cDNAsequence included in the deposited clone, which is hybridizable to SEQID NO:1.

The invention further relates to a polynucleotide encoding a polypeptideof SEQ ID NO:2 or the cDNA sequence included in the deposited clone,which is hybridizable to SEQ ID NO:1, having biological activity.

The invention further relates to a polynucleotide which is a variant ofSEQ ID NO:1.

The invention further relates to a polynucleotide which is an allelicvariant of SEQ ID NO:1.

The invention further relates to a polynucleotide which encodes aspecies homologue of the SEQ ID NO:2.

The invention further relates to a polynucleotide which represents thecomplimentary sequence (antisense) of SEQ ID NO:1.

The invention further relates to a polynucleotide capable of hybridizingunder stringent conditions to any one of the polynucleotides specifiedherein, wherein said polynucleotide does not hybridize under stringentconditions to a nucleic acid molecule having a nucleotide sequence ofonly A residues or of only T residues.

The invention further relates to an isolated nucleic acid molecule ofSEQ ID NO:2, wherein the polynucleotide fragment comprises a nucleotidesequence encoding a leucine-rich repeat protein.

The invention further relates to an isolated nucleic acid molecule ofSEQ ID NO:1, 3, or 5 wherein the polynucleotide fragment comprises anucleotide sequence encoding the sequence identified as SEQ ID NO:2 orthe polypeptide encoded by the cDNA sequence included in the depositedclone, which is hybridizable to SEQ ID NO:1.

The invention further relates to an isolated nucleic acid molecule of ofSEQ ID NO:1, 3, or 5, wherein the polynucleotide fragment comprises theentire nucleotide sequence of SEQ ID NO:1 or the cDNA sequence includedin the deposited clone, which is hybridizable to SEQ ID NO:1.

The invention further relates to an isolated nucleic acid molecule ofSEQ ID NO:1, wherein the nucleotide sequence comprises sequentialnucleotide deletions from either the C-terminus or the N-terminus.

The invention further relates to an isolated polypeptide comprising anamino acid sequence that comprises a polypeptide fragment of SEQ ID NO:2or the encoded sequence included in the deposited clone.

The invention further relates to a polypeptide fragment of SEQ ID NO:2or the encoded sequence included in the deposited clone, havingbiological activity.

The invention further relates to a polypeptide domain of SEQ ID NO:2 orthe encoded sequence included in the deposited clone.

The invention further relates to a polypeptide epitope of SEQ ID NO:2 orthe encoded sequence included in the deposited clone.

The invention further relates to a full length protein of SEQ ID NO:2 orthe encoded sequence included in the deposited clone.

The invention further relates to a variant of SEQ ID NO:2.

The invention further relates to an allelic variant of SEQ ID NO:2. Theinvention further relates to a species homologue of SEQ ID NO:2.

The invention further relates to the isolated polypeptide of of SEQ IDNO:2, wherein the full length protein comprises sequential amino aciddeletions from either the C-terminus or the N-terminus.

The invention further relates to an isolated antibody that bindsspecifically to the isolated polypeptide of SEQ ID NO:2.

The invention further relates to a method for preventing, treating, orameliorating a medical condition, comprising administering to amammalian subject a therapeutically effective amount of the polypeptideof SEQ ID NO:2 or the polynucleotide of SEQ ID NO:1.

The invention further relates to a method of diagnosing a pathologicalcondition or a susceptibility to a pathological condition in a subjectcomprising the steps of (a) determining the presence or absence of amutation in the polynucleotide of SEQ ID NO:1; and (b) diagnosing apathological condition or a susceptibility to a pathological conditionbased on the presence or absence of said mutation.

The invention further relates to a method of diagnosing a pathologicalcondition or a susceptibility to a pathological condition in a subjectcomprising the steps of (a) determining the presence or amount ofexpression of the polypeptide of of SEQ ID NO:2 in a biological sample;and diagnosing a pathological condition or a susceptibility to apathological condition based on the presence or amount of expression ofthe polypeptide.

The invention further relates to a method for identifying a bindingpartner to the polypeptide of SEQ ID NO:2 comprising the steps of (a)contacting the polypeptide of SEQ ID NO:2 with a binding partner; and(b) determining whether the binding partner effects an activity of thepolypeptide.

The invention further relates to a gene corresponding to the cDNAsequence of SEQ ID NO:1.

The invention further relates to a method of identifying an activity ina biological assay, wherein the method comprises the steps of expressingSEQ ID NO:1 in a cell, (b) isolating the supernatant; (c) detecting anactivity in a biological assay; and (d) identifying the protein in thesupernatant having the activity.

The invention further relates to a process for making polynucleotidesequences encoding gene products having altered activity selected fromthe group consisting of SEQ ID NO:2 activity comprising the steps of (a)shuffling a nucleotide sequence of SEQ ID NO:1, (b) expressing theresulting shuffled nucleotide sequences and, (c) selecting for alteredactivity selected from the group consisting of SEQ ID NO:2 activity ascompared to the activity selected from the group consisting of SEQ IDNO:2 activity of the gene product of said unmodified nucleotidesequence.

The invention further relates to a shuffled polynucleotide sequenceproduced by a shuffling process, wherein said shuffled DNA moleculeencodes a gene product having enhanced tolerance to an inhibitor of anyone of the activities selected from the group consisting of SEQ ID NO:2activity.

The invention further relates to a method for preventing, treating, orameliorating a medical condition with the polypeptide provided as SEQ IDNO:2, in addition to, its encoding nucleic acid, wherein the medicalcondition is a cardiovascular disorder

The invention further relates to a method for preventing, treating, orameliorating a medical condition with the polypeptide provided as SEQ IDNO:2, in addition to, its encoding nucleic acid, wherein the medicalcondition is a reproductive disorder.

The invention further relates to a method for preventing, treating, orameliorating a medical condition with the polypeptide provided as SEQ IDNO:2, in addition to, its encoding nucleic acid, wherein the medicalcondition is a neural disorder.

The invention further relates to a method for preventing, treating, orameliorating a medical condition, wherein the medical condition isdisorder related to aberrant apoptosis modulation, either directly orindirectly.

The invention further relates to a method for preventing, treating, orameliorating a medical condition, wherein the medical condition is adisorder related to aberrant cell adhesion, either directly orindirectly.

The invention further relates to a method for preventing, treating, orameliorating a medical condition, wherein the medical condition is adisorder related to aberrant cell matrix organization, either directlyor indirectly.

The invention further relates to a method for preventing, treating, orameliorating a medical condition, wherein the medical condition is acancer.

The invention further relates to a method of identifying a compound thatmodulates the biological activity of HLRRCR-1, comprising the steps of,(a) combining a candidate modulator compound with HLRRCR-1 having thesequence set forth in one or more of SEQ ID NO:2 or 4; and measuring aneffect of the candidate modulator compound on the activity of HLRRCR-1.

The invention further relates to a method of identifying a compound thatmodulates the biological activity of a leucine-rich repeat protein,comprising the steps of, (a) combining a candidate modulator compoundwith a host cell expressing HLRRCR-1 having the sequence as set forth inSEQ ID NOS:2 or 4; and, (b) measuring an effect of the candidatemodulator compound on the activity of the expressed HLRRCR-1.

The invention further relates to a method of identifying a compound thatmodulates the biological activity of HLRRCR-1, comprising the steps of,(a) combining a candidate modulator compound with a host cell containinga vector described herein, wherein HLRRCR-1 is expressed by the cell;and, (b) measuring an effect of the candidate modulator compound on theactivity of the expressed HLRRCR-1.

The invention further relates to a method of screening for a compoundthat is capable of modulating the biological activity of HLRRCR-1,comprising the steps of: (a) providing a host cell described herein; (b)determining the biological activity of HLRRCR-1 in the absence of amodulator compound; (c) contacting the cell with the modulator compound;and (d) determining the biological activity of HLRRCR-1 in the presenceof the modulator compound; wherein a difference between the activity ofHLRRCR-1 in the presence of the modulator compound and in the absence ofthe modulator compound indicates a modulating effect of the compound.

The invention further relates to a compound that modulates thebiological activity of human HLRRCR-1 as identified by the methodsdescribed herein.

The invention further relates to a method for preventing, treating, orameliorating a medical condition with the polypeptide provided as SEQ IDNO:2, in addition to, its encoding nucleic acid, or a modulator thereof,wherein the medical condition is a member of the group consisting of:lung cancer, related proliferative condition of the lung, colon cancer,and related proliferative condition of the colon.

The invention further relates to a method of diagnosing a pathologicalcondition or a susceptibility to a pathological condition in a subjectcomprising the steps of (a) determining the presence or amount ofexpression of the polypeptide of of SEQ ID NO:2 in a biological sample;and diagnosing a pathological condition or a susceptibility to apathological condition based on the presence or amount of expression ofthe polypeptide, wherein said condition is a member of the groupconsisting of lung cancer, proliferative condition of the lung, ovariancancer, colon cancer, leukemias, and melanoma.

The invention further relates to a method for preventing, treating, orameliorating a medical condition with the polypeptide provided as SEQ IDNO:2, in addition to, its encoding nucleic acid, or a modulator thereof,wherein the medical condition is neural disorders, memory disorders,establishment of short term memory, establishment of long term memory,dimentia, cognition, learning, the development of long termpotentiation, disorders associated with neuron attrition, Alzheimers,Parkinson's, disorders that accompany sniffing of volatile vapors,disorders that accompany severe trauma to the brain, in addition toother disorders associated with the brain, particularly memorydisorders.

The invention further relates to a method of diagnosing a pathologicalcondition or a susceptibility to a pathological condition in a subjectcomprising the steps of (a) determining the presence or amount ofexpression of the polypeptide of of SEQ ID NO:2 in a biological sample;(b) and diagnosing a pathological condition or a susceptibility to apathological condition based on the presence or amount of expression ofthe polypeptide relative to a control, wherein said condition is amember of the group consisting of movement disorders, such asParkinson's, other forms of ataxia, neural disorders, memory disorders,establishment of short term memory, establishment of long term memory,dimentia, cognition, learning, the development of long termpotentiation, disorders associated with neuron attrition, Alzheimers,Parkinson's, disorders that accompany sniffing of volatile vapors,disorders that accompany severe trauma to the brain, in addition toother disorders associated with the brain, particularly memorydisorders.

BRIEF DESCRIPTION OF THE FIGURES/DRAWINGS

FIGS. 1A-C show the polynucleotide sequence (SEQ ID NO:1) and deducedamino acid sequence (SEQ ID NO:2) of the novel human leucine-rich repeatcontaining protein, HLLRCR-1, of the present invention. The standardone-letter abbreviation for amino acids is used to illustrate thededuced amino acid sequence. The polynucleotide sequence contains asequence of 1874 nucleotides (SEQ ID NO:1), encoding a polypeptide of592 amino acids (SEQ ID NO:2). An analysis of the HLLRCR-1 polypeptidedetermined that it comprised the following features: a predictedtransmembrane domain located from about amino acid 291 to about aminoacid 312 of SEQ ID NO:2 represented by double underlining; six conservedleucine rich repeat domains located from about amino acid 58 to aboutamino acid 81, from about amino acid 82 to about amino acid 107, fromabout amino acid 108 to about amino acid 131, from about amino acid 132to about amino acid 155, from about amino acid 158 to about amino acid181, and/or from about amino acid 182 to about amino acid 204 of SEQ IDNO:2 represented by light shading; and a conserved leucine-rich repeatC-terminal domain located from about amino acid 214 to about amino acid270 of SEQ ID NO:2 represented by dark shading; conserved cysteineresidues located at amino acid 11, 217, 219, 247, 307 and/or 425 of SEQID NO:2 represented in bold; and conserved leucine residues located atamino acid 4, 25, 37, 45, 55, 58, 61, 63, 87, 97, 106, 108, 111, 113,122, 132, 135, 137, 146, 155, 158, 161, 163, 179, 192, 187, 192, 203,206, 208, 210, 225, 228, 264, 277, 306, 317, 357, 402, 419, 439, 451,454, 465, 490, 495, and/or 499 of SEQ ID NO:2 represented by singleunderlining. The conserved leucine residues are characteristic ofleucine-rich repeat proteins as described more particularly elsewhereherein. The conserved cysteine residues are diagnostic of conservedstructural features of the protein to leucine-rich repeat containingproteins (particularly those referenced herein), and may be indicativeof conserved protein function.

FIGS. 2A-C show the regions of identity between the encoded HLLRCR-1protein (SEQ ID NO:2) to other leucine-rich repeat proteins,specifically, the human NoGo receptor protein (Human_NOGOR; GenbankAccession No:gi|13647496; SEQ ID NO:6); the mouse NoGo receptor protein(Mouse_NogoR; Genbank Accession No:gi|12407651; SEQ ID NO:8); thecrab-eating macaque brain protein 1 (Macaca_brainprotein1; GenbankAccession No:gi|12698137; SEQ ID NO:5); the crab-eating macaque brainprotein 2 (Macaca_brainprotein2; Genbank Accession No:gi|9280025; SEQ IDNO:7); the Drosophila CG7509 protein (Fly_CG7509; Genbank AccessionNo:gi|7292539; SEQ ID NO:9); and the mouse orphan G protein-coupledreceptor FEX protein (mouse_GPCRFEX; Genbank Accession No:gi|6753842;SEQ ID NO:10). The alignment was performed using the CLUSTALW algorithmdescribed elsewhere herein. Lines between residues indicate gappedregions of non-identity for the aligned polypeptides, asterisks belowthe aligned polypeptides indicate identical amino acids for all of thealigned polypeptides, double dots indicate identical amino aicds forless than all of the aligned polypeptides, and single dots indicatesimilar amino acid residues. The conserved leucine residues betweenHLLRCR-1 and the other leucine-rich repeat containing proteins arenoted.

FIG. 3 shows the regions of local identity and similarity between theencoded HLLRCR-1 protein (SEQ ID NO:2) to the human Pfam Leucine richrepeat C-terminal domain consensus sequence (LRRCT; Pfam Accession No:PF01463). The query (“Q”) sequence represents the local matchingsequence of the HLLRCR-1 protein (SEQ ID NO:2), whereas the target (“T”)represents the human Pfam Leucine rich repeat C-terminal domainconsensus sequence. The alignment was performed using the BLAST2algorithm according to default parameters (S F Altschul, et al., NucleicAcids Res 25:3389-3402, 1997). The amino acids between the query andtarget sequences represent matching identical amino acids between thetwo sequences. Plus signs (“+”) between the query and target sequencesrepresent similar amino acids between the two sequences. Dots (“•”)between the query and target sequences indicate regions of non-identityfor the aligned polypeptides. The conserved leucines between HLLRCR-1and the consensus Leucine rich repeat C-terminal domain polypeptidesequence are noted and described herein.

FIG. 4 shows the regions of local identity and similarity between theencoded HLLRCR-1 protein (SEQ ID NO:2) to the human Pfam Leucine RichRepeat consensus sequence (LRR; Pfam Accession No: PF00560). The query(“Q”) sequence represents the local matching sequence of the HLLRCR-1protein (SEQ ID NO:2), whereas the target (“T”) represents the humanPfam LRR consensus sequence. The alignment was performed using theBLAST2 algorithm according to default parameters (S F Altschul, et al.,Nucleic Acids Res 25:3389-3402, 1997). The amino acids between the queryand target sequences represent matching identical amino acids betweenthe two sequences. Plus signs (“+”) between the query and targetsequences represent similar amino acids between the two sequences. Dots(“•”) between the query and target sequences indicate regions ofnon-identity for the aligned polypeptides. The conserved leucinesbetween HLLRCR-1 and the consensus LRR polypeptide sequence are notedand described herein.

FIG. 5 shows an expression profile of the novel leucine-rich repeatcontaining protein, HLLRCR-1. The figure illustrates the relativeexpression level of HLLRCR-1 amongst various mRNA tissue sources. Asshown, transcripts corresponding to HLLRCR-1 expressed predominatelyhigh in the heart; significantly in testis; and to a lesser extent, inbrain, spinal cord, and brain subregions. Expression data was obtainedby measuring the steady state HLLRCR-1 mRNA levels by quantitative PCRusing the PCR primer pair provided as SEQ ID NO:20 and 21 as describedherein. Brain subregion abbreviations are as follows: A=amygdala;C=cerebellum; CC=corpus callosum; CN=caudate nucleus; H=hippocampus;SN=substantia nigra; and T=thalamus.

FIG. 6 shows a table illustrating the percent identity and percentsimilarity values between the full-length HLLRCR-1 polypeptide of thepresent invention with other leucine-rich repeat containing proteins,specifically, the human NoGo receptor protein (Human_NOGOR; GenbankAccession No:gi|13647496; SEQ ID NO:6); the mouse NoGo receptor protein(Mouse_NogoR; Genbank Accession No:gi|12407651; SEQ ID NO:8); thecrab-eating macaque brain protein 1 (Macaca_brainprotein1; GenbankAccession No:gi|12698137; SEQ ID NO:5); the crab-eating macaque brainprotein 2 (Macaca_brainprotein2; Genbank Accession No:gi|9280025; SEQ IDNO:7); the Drosophila CG7509 protein (Fly_CG7509; Genbank AccessionNo:gi|7292539; SEQ ID NO:9); and the mouse orphan G protein-coupledreceptor FEX protein (mouse_GPCRFEX; Genbank Accession No:gi|6753842;SEQ ID NO:10). The percent identity and percent similarity values weredetermined using the BLAST algorithm using default parameters (S FAltschul, T L Madden, A A Schaffer, J Zhang, Z Zhang, W Miller, D JLipman. Gapped BLAST and PSI-BLAST: a new generation of protein databasesearch programs. Nucleic Acids Res 25:3389-3402, 1997).

FIG. 7 shows the polynucleotide and polypeptide sequence of a portion ofhuman bac AL139285. The polynucleotide sequence of bac AL139285 (SEQ IDNO:45) was used to design oligonucleotide primers to clone the humanHLRRCR-1 gene of the present invention, as described elsewhere herein.

FIG. 8 shows an expanded expression profile of the novel human G-proteincoupled receptor, HLRRCR-1, of the present invention. The figureillustrates the relative expression level of HLRRCR-1 amongst mRNAisolated from a number of cancer cell lines. As shown, the HLRRCR-1polypeptide was expressed predominately in several lung cancer celllines, significantly in a colon tumor cell lines, and to a lesser extentin other human tumor cell lines as shown. The results suggest HLRRCR-1may be diagnostic of transformed phenotypes of the lung and coloncancers. Expression data was obtained by measuring the steady stateHLRRCR-1 mRNA levels by quantitative PCR using the PCR primer pairprovided as SEQ ID NO:89 and 90 as described in Example 5 herein.

FIG. 9 shows an expanded expression profile of the novel human G-proteincoupled receptor, HLRRCR-1. The figure illustrates the relativeexpression level of HLRRCR-1 amongst various mRNA tissue sourcesisolated from normal tissues. As shown, the HLRRCR-1 polypeptide wasexpressed predominately in hippocampus and throughout the cortex of thebrain. HLRRCR-1 was also significant expressed in the left ventricle ofthe heart, the DRG, and to a lesser extent in other tissues as shown.Expression data was obtained by measuring the steady state HLRRCR-1 mRNAlevels by quantitative PCR using the PCR primer pair provided as SEQ IDNO:94 and 95, and Taqman probe (SEQ ID NO:96) as described in Example 6herein.

FIG. 10 shows an expanded expression profile of the novel humanG-protein coupled receptor, HLRRCR-1, of the present invention. Thefigure illustrates the relative expression level of HLRRCR-1 amongstvarious mRNA tissue sources isolated from normal and tumor tissues. Asshown, the HLRRCR-1 polypeptide was differentially expressed inParkinson's substantia nigra tissue compared to each respective normaltissue. Expression data was obtained by measuring the steady stateHLRRCR-1 mRNA levels by quantitative PCR using the PCR primer pairprovided as SEQ ID NO:94 and 95, and Taqman probe (SEQ ID NO:96) asdescribed in Example 6 herein.

Table I provides a summary of the novel polypeptides and their encodingpolynucleotides of the present invention.

Table II illustrates the preferred hybridization conditions for thepolynucleotides of the present invention. Other hybridization conditionsmay be known in the art or are described elsewhere herein.

Table III provides a summary of various conservative substitutionsencompassed by the present invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention may be understood more readily by reference to thefollowing detailed description of the preferred embodiments of theinvention and the Examples included herein.

The invention provides a novel human sequence that encodes a novelleucine-rich repeat containing protein, HLLRCR-1, with substantialhomology to the leucine-rich repeat containing protein known as the NoGoreceptor. The NoGo receptor is primarily expressed in the brain and isthought to be responsible for modulating neurite growth. Expressionanalysis indicates the HLLRCR-1 has strong preferential expression inthe heart; significantly in testis; and to a lesser extent, in brain,spinal cord, and brain subregions. Based on this information, we haveprovisionally named the gene and protein HLLRCR-1 (Human Leucine-RichRepeat Cardiac Receptor-1). The specificity by which the HLLRCR-1transcript is expressed suggests its importance in various biologicalprocesses. The polynucleotide and polypeptide of the present inventionis sometimes referred to herein as HLLRCR-1, HLLRCR-1b, or full-lengthHLLRCR-1. Unless otherwise noted, HLLRCR-1 nomenclature applies to boththe HLLRCR-1b, and full-length HLLRCR-1 polynucleotide and polypeptide.

In the present invention, “isolated” refers to material removed from itsoriginal environment (e.g., the natural environment if it is naturallyoccurring), and thus is altered “by the hand of man” from its naturalstate. For example, an isolated polynucleotide could be part of a vectoror a composition of matter, or could be contained within a cell, andstill be “isolated” because that vector, composition of matter, orparticular cell is not the original environment of the polynucleotide.The term “isolated” does not refer to genomic or cDNA libraries, wholecell total or mRNA preparations, genomic DNA preparations (includingthose separated by electrophoresis and transferred onto blots), shearedwhole cell genomic DNA preparations or other compositions where the artdemonstrates no distinguishing features of the polynucleotide/sequencesof the present invention.

In specific embodiments, the polynucleotides of the invention are atleast 15, at least 30, at least 50, at least 100, at least 125, at least500, or at least 1000 continuous nucleotides but are less than or equalto 300 kb, 200 kb, 100 kb, 50 kb, 15 kb, 10 kb, 7.5 kb, 5 kb, 2.5 kb,2.0 kb, or 1 kb, in length. In a further embodiment, polynucleotides ofthe invention comprise a portion of the coding sequences, as disclosedherein, but do not comprise all or a portion of any intron. In anotherembodiment, the polynucleotides comprising coding sequences do notcontain coding sequences of a genomic flanking gene (i.e., 5′ or 3′ tothe gene of interest in the genome). In other embodiments, thepolynucleotides of the invention do not contain the coding sequence ofmore than 1000, 500, 250, 100, 50, 25, 20, 15, 10, 5, 4, 3, 2, or 1genomic flanking gene(s).

As used herein, a “polynucleotide” refers to a molecule having a nucleicacid sequence contained in SEQ ID NO:1, SEQ ID NO:3, or the cDNAcontained within the clone deposited with the ATCC. For example, thepolynucleotide can contain the nucleotide sequence of the full lengthcDNA sequence, including the 5′ and 3′ untranslated sequences, thecoding region, with or without a signal sequence, the secreted proteincoding region, as well as fragments, epitopes, domains, and variants ofthe nucleic acid sequence. Moreover, as used herein, a “polypeptide”refers to a molecule having the translated amino acid sequence generatedfrom the polynucleotide as broadly defined.

In the present invention, the full length sequence identified as SEQ IDNO:1 and SEQ ID NO:3, respectfully, was often generated by overlappingsequences contained in one or more clones (contig analysis). Arepresentative clone containing all or most of the sequence for SEQ IDNO:1 and SEQ ID NO:3 was deposited with the American Type CultureCollection (“ATCC”). As shown in Table I, each clone is identified by acDNA Clone ID (Identifier) and the ATCC Deposit Number. The ATCC islocated at 10801 University Boulevard, Manassas, Va. 20110-2209, USA.The ATCC deposit was made pursuant to the terms of the Budapest Treatyon the international recognition of the deposit of microorganisms forpurposes of patent procedure. The deposited clone is inserted in thepSport1 plasmid (Life Technologies) using SalI and NotI restrictionsites described herein.

Unless otherwise indicated, all nucleotide sequences determined bysequencing a DNA molecule herein were determined using an automated DNAsequencer (such as the Model 373, preferably a Model 3700, from AppliedBiosystems, Inc.), and all amino acid sequences of polypeptides encodedby DNA molecules determined herein were predicted by translation of aDNA sequence determined above. Therefore, as is known in the art for anyDNA sequence determined by this automated approach, any nucleotidesequence determined herein may contain some errors. Nucleotide sequencesdetermined by automation are typically at least about 90% identical,more typically at least about 95% to at least about 99.9% identical tothe actual nucleotide sequence of the sequenced DNA molecule. The actualsequence can be more precisely determined by other approaches includingmanual DNA sequencing methods well known in the art. As is also known inthe art, a single insertion or deletion in a determined nucleotidesequence compared to the actual sequence will cause a frame shift intranslation of the nucleotide sequence such that the predicted aminoacid sequence encoded by a determined nucleotide sequence will becompletely different from the amino acid sequence actually encoded bythe sequenced DNA molecule, beginning at the point of such an insertionor deletion.

Using the information provided herein, such as the nucleotide sequencein FIGS. 1A-C (SEQ ID NO:1), a nucleic acid molecule of the presentinvention encoding the HLLRCR-1 polypeptide may be obtained usingstandard cloning and screening procedures, such as those for cloningcDNAs using mRNA as starting material. Illustrative of the invention,the nucleic acid molecule described in FIGS. 1A-C (SEQ ID NO:1), wasdiscovered in a human brain and testis cDNA library.

A “polynucleotide” of the present invention also includes thosepolynucleotides capable of hybridizing, under stringent hybridizationconditions, to sequences contained in SEQ ID NO:1, or the complementthereof; in SEQ ID NO:3, or the complement thereof; or the cDNA withinthe clone deposited with the ATCC. “Stringent hybridization conditions”refers to an overnight incubation at 42 degree C. in a solutioncomprising 50% formamide, 5×SSC (750 mM NaCl, 75 mM trisodium citrate),50 mM sodium phosphate (pH 7.6), 5× Denhardt's solution, 10% dextransulfate, and 20 μg/ml denatured, sheared salmon sperm DNA, followed bywashing the filters in 0.1× SSC at about 65 degree C.

Also contemplated are nucleic acid molecules that hybridize to thepolynucleotides of the present invention at lower stringencyhybridization conditions. Changes in the stringency of hybridization andsignal detection are primarily accomplished through the manipulation offormamide concentration (lower percentages of formamide result inlowered stringency); salt conditions, or temperature. For example, lowerstringency conditions include an overnight incubation at 37 degree C. ina solution comprising 6×SSPE (20×SSPE=3M NaCl; 0.2M NaH2PO4; 0.02M EDTA,pH 7.4), 0.5% SDS, 30% formamide, 100 ug/ml salmon sperm blocking DNA;followed by washes at 50 degree C. with 1×SSPE, 0.1% SDS. In addition,to achieve even lower stringency, washes performed following stringenthybridization can be done at higher salt concentrations (e.g. 5×SSC).

Note that variations in the above conditions may be accomplished throughthe inclusion and/or substitution of alternate blocking reagents used tosuppress background in hybridization experiments. Typical blockingreagents include Denhardt's reagent, BLOTTO, heparin, denatured salmonsperm DNA, and commercially available proprietary formulations. Theinclusion of specific blocking reagents may require modification of thehybridization conditions described above, due to problems withcompatibility.

Of course, a polynucleotide which hybridizes only to polyA+ sequences(such as any 3′ terminal polyA+ tract of a cDNA shown in the sequencelisting), or to a complementary stretch of T (or U) residues, would notbe included in the definition of “polynucleotide,” since such apolynucleotide would hybridize to any nucleic acid molecule containing apoly (A) stretch or the complement thereof (e.g., practically anydouble-stranded cDNA clone generated using oligo dT as a primer).

The polynucleotide of the present invention can be composed of anypolyribonucleotide or polydeoxribonucleotide, which may be unmodifiedRNA or DNA or modified RNA or DNA. For example, polynucleotides can becomposed of single- and double-stranded DNA, DNA that is a mixture ofsingle- and double-stranded regions, single- and double-stranded RNA,and RNA that is mixture of single- and double-stranded regions, hybridmolecules comprising DNA and RNA that may be single-stranded or, moretypically, double-stranded or a mixture of single- and double-strandedregions. In addition, the polynucleotide can be composed oftriple-stranded regions comprising RNA or DNA or both RNA and DNA. Apolynucleotide may also contain one or more modified bases or DNA or RNAbackbones modified for stability or for other reasons. “Modified” basesinclude, for example, tritylated bases and unusual bases such asinosine. A variety of modifications can be made to DNA and RNA; thus,“polynucleotide” embraces chemically, enzymatically, or metabolicallymodified forms.

The polypeptide of the present invention can be composed of amino acidsjoined to each other by peptide bonds or modified peptide bonds, i.e.,peptide isosteres, and may contain amino acids other than the 20gene-encoded amino acids. The polypeptides may be modified by eithernatural processes, such as posttranslational processing, or by chemicalmodification techniques which are well known in the art. Suchmodifications are well described in basic texts and in more detailedmonographs, as well as in a voluminous research literature.Modifications can occur anywhere in a polypeptide, including the peptidebackbone, the amino acid side-chains and the amino or carboxyl termini.It will be appreciated that the same type of modification may be presentin the same or varying degrees at several sites in a given polypeptide.Also, a given polypeptide may contain many types of modifications.Polypeptides may be branched, for example, as a result ofubiquitination, and they may be cyclic, with or without branching.Cyclic, branched, and branched cyclic polypeptides may result fromposttranslation natural processes or may be made by synthetic methods.Modifications include acetylation, acylation, ADP-ribosylation,amidation, covalent attachment of flavin, covalent attachment of a hememoiety, covalent attachment of a nucleotide or nucleotide derivative,covalent attachment of a lipid or lipid derivative, covalent attachmentof phosphotidylinositol, cross-linking, cyclization, disulfide bondformation, demethylation, formation of covalent cross-links, formationof cysteine, formation of pyroglutamate, formylation,gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation,iodination, methylation, myristoylation, oxidation, pegylation,proteolytic processing, phosphorylation, prenylation, racemization,selenoylation, sulfation, transfer-RNA mediated addition of amino acidsto proteins such as arginylation, and ubiquitination. (See, forinstance, Proteins—Structure and Molecular Properties, 2nd Ed., T. E.Creighton, W. H. Freeman and Company, New York (1993); PosttranslationalCovalent Modification of Proteins, B. C. Johnson, Ed., Academic Press,New York, pgs. 1-12 (1983); Seifter et al., Meth Enzymol 182:626-646(1990); Rattan et al., Ann NY Acad Sci 663:48-62 (1992).)

“SEQ ID NO:1” refers to a polynucleotide sequence while “SEQ ID NO:2”refers to a polypeptide sequence, both sequences are identified by aninteger specified in Table I.

“SEQ ID NO:3” refers to a polynucleotide sequence while “SEQ ID NO:4”refers to a polypeptide sequence, both sequences are identified by aninteger specified in Table I.

“A polypeptide having biological activity” refers to polypeptidesexhibiting activity similar, but not necessarily identical to, anactivity of a polypeptide of the present invention, including matureforms, as measured in a particular biological assay, with or withoutdose dependency. In the case where dose dependency does exist, it neednot be identical to that of the polypeptide, but rather substantiallysimilar to the dose-dependence in a given activity as compared to thepolypeptide of the present invention (i.e., the candidate polypeptidewill exhibit greater activity or not more than about 25-fold less and,preferably, not more than about tenfold less activity, and mostpreferably, not more than about three-fold less activity relative to thepolypeptide of the present invention.)

It is another aspect of the present invention to provide modulators ofthe HLLRCR-1 protein and HLLRCR-1 peptide targets which can affect thefunction or activity of HLLRCR-1 in a cell in which HLLRCR-1 function oractivity is to be modulated or affected. In addition, modulators ofHLLRCR-1 can affect downstream systems and molecules that are regulatedby, or which interact with, HLLRCR-1 in the cell. Modulators of HLLRCR-1include compounds, materials, agents, drugs, and the like, thatantagonize, inhibit, reduce, block, suppress, diminish, decrease, oreliminate HLLRCR-1 function and/or activity. Such compounds, materials,agents, drugs and the like can be collectively termed “antagonists”.Alternatively, modulators of HLLRCR-1 include compounds, materials,agents, drugs, and the like, that agonize, enhance, increase, augment,or amplify HLLRCR-1 function in a cell. Such compounds, materials,agents, drugs and the like can be collectively termed “agonists”.

As used herein the terms “modulate” or “modulates” refer to an increaseor decrease in the amount, quality or effect of a particular activity,DNA, RNA, or protein. The definition of “modulate” or “modulates” asused herein is meant to encompass agonists and/or antagonists of aparticular activity, DNA, RNA, or protein.

As will be appreciated by the skilled practitioner, should the aminoacid fragment comprise an antigenic epitope, for example, biologicalfunction per se need not be maintained. The terms HLLRCR-1 polypeptideand HLLRCR-1 protein are used interchangeably herein to refer to theencoded product of the HLLRCR-1 nucleic acid sequence according to thepresent invention.

The term “organism” as referred to herein is meant to encompass anyorganism referenced herein, though preferably to eukaryotic organisms,more preferably to mammals, and most preferably to humans.

The present invention encompasses the identification of proteins,nucleic acids, or other molecules, that bind to polypeptides andpolynucleotides of the present invention (for example, in areceptor-ligand interaction). The polynucleotides of the presentinvention can also be used in interaction trap assays (such as, forexample, that described by Ozenberger and Young (Mol Endocrinol.,9(10):1321-9, (1995); and Ann. N. Y. Acad. Sci., 7;766:279-81, (1995)).

The polynucleotide and polypeptides of the present invention are usefulas probes for the identification and isolation of full-length cDNAsand/or genomic DNA which correspond to the polynucleotides of thepresent invention, as probes to hybridize and discover novel, relatedDNA sequences, as probes for positional cloning of this or a relatedsequence, as probe to “subtract-out” known sequences in the process ofdiscovering other novel polynucleotides, as probes to quantify geneexpression, and as probes for microarrays.

In addition, polynucleotides and polypeptides of the present inventionmay comprise one, two, three, four, five, six, seven, eight, or moremembrane domains.

Also, in preferred embodiments the present invention provides methodsfor further refining the biological function of the polynucleotidesand/or polypeptides of the present invention.

Specifically, the invention provides methods for using thepolynucleotides and polypeptides of the invention to identify orthologs,homologs, paralogs, variants, and/or allelic variants of the invention.Also provided are methods of using the polynucleotides and polypeptidesof the invention to identify the entire coding region of the invention,non-coding regions of the invention, regulatory sequences of theinvention, and secreted, mature, pro-, prepro-, forms of the invention(as applicable).

In preferred embodiments, the invention provides methods for identifyingthe glycosylation sites inherent in the polynucleotides and polypeptidesof the invention, and the subsequent alteration, deletion, and/oraddition of said sites for a number of desirable characteristics whichinclude, but are not limited to, augmentation of protein folding,inhibition of protein aggregation, regulation of intracellulartrafficking to organelles, increasing resistance to proteolysis,modulation of protein antigenicity, and mediation of intercellularadhesion.

In further preferred embodiments, methods are provided for evolving thepolynucleotides and polypeptides of the present invention usingmolecular evolution techniques in an effort to create and identify novelvariants with desired structural, functional, and/or physicalcharacteristics.

The present invention further provides for other experimental methodsand procedures currently available to derive functional assignments.These procedures include but are not limited to spotting of clones onarrays, micro-array technology, PCR based methods (e.g., quantitativePCR), anti-sense methodology, gene knockout experiments, and otherprocedures that could use sequence information from clones to build aprimer or a hybrid partner.

Polynucleotides and Polypeptides of the Invention

Features of the Polypeptide Encoded by Gene No: 1

The partial polypeptide of this gene provided as SEQ ID NO:2 (FIGS.1A-C), encoded by the polynucleotide sequence according to SEQ ID NO:1(FIGS. 1A-C), and/or encoded by the polynucleotide contained within thedeposited clone, HLLRCR-1 (also refered to as GPCR111, GPCR111-80b,and/or HLLRCAR-1), has significant homology at the nucleotide and aminoacid level to a number of leucine-rich repeat containing proteins, whichinclude, for example, human NoGo receptor protein (Human_NOGOR; GenbankAccession No:gi|13647496; SEQ ID NO:6); the mouse NoGo receptor protein(Mouse_NogoR; Genbank Accession No:gi|12407651; SEQ ID NO:8); thecrab-eating macaque brain protein 1 (Macaca_brainprotein1; GenbankAccession No:gi|12698137; SEQ ID NO:5); the crab-eating macaque brainprotein 2 (Macaca_brainprotein2; Genbank Accession No:gi|9280025; SEQ IDNO:7); the Drosophila CG7509 protein (Fly_CG7509; Genbank AccessionNo:gi|7292539; SEQ ID NO:9); and the mouse orphan G protein-coupledreceptor FEX protein (mouse_GPCRFEX; Genbank Accession No:gi|6753842;SEQ ID NO:10). An alignment of the HLLRCR-1 polypeptide with theseproteins is provided in FIGS. 2A-C.

The determined nucleotide sequence of the full-length HLLRCR-1 cDNA inFIGS. 1A-C (SEQ ID NO:1) contains an open reading frame encoding aprotein of about 592 amino acid residues, with a deduced molecularweight of about 66.4 kDa. The amino acid sequence of the predictedfull-length HLLRCR-1 polypeptide is shown in FIGS. 1A-C (SEQ ID NO:2).The full-length HLLRCR-1 protein shown in FIGS. 1A-C was determined tobe about 28.1% identical and 33.4% similar to the human NoGo receptorprotein (Human_NOGOR; Genbank Accession No:gi|13647496; SEQ ID NO:6); tobe about 27.8% identical and 32.6% similar to the mouse NoGo receptorprotein (Mouse_NogoR; Genbank Accession No:gi|12407651; SEQ ID NO:8); tobe about 93.7% identical and 94.3% similar to the crab-eating macaquebrain protein 1 (Macaca_brainprotein1; Genbank Accession No:gi|12698137;SEQ ID NO:5); to be about 28.7% identical and 34.1% similar to thecrab-eating macaque brain protein 2 (Macaca_brainprotein2; GenbankAccession No;:gi|9280025; SEQ ID NO:7); to be about 26.9% identical and37.0% similar to the Drosophila CG7509 protein (Fly_CG7509; GenbankAccession No:gi|7292539; SEQ ID NO:9); and to be about 30.7% identicaland 37.6% similar to the mouse orphan G protein-coupled receptor FEXprotein (mouse_GPCRFEX; Genbank Accession No:gi|6753842; SEQ ID NO:10),as shown in FIG. 6.

The human NoGo receptor protein (Human_NOGOR; Genbank AccessionNo:gi|13647496; SEQ ID NO:6) a novel myelin-associated inhibitor ofneurite outgrowth which regulates stable neuronal connections duringaxonal regeneration following injury in the adult mammalian centralnervous system (CNS). Recent evidence suggests that the NoGo receptorinhibits neurite outgrowth and that antagonizing the activty of the NoGoreceptor, and/or its upstream or downstream affectors may provide anopportunity for neurons to regenerate post injury (Nature,27;417(6892):941-4 (2002; Neuropathol Appl Neurobiol. 28(2):95-106(2002)). In addition, there is increasing evidence that Nogo expressionmay induce apoptosis in tumor cells and be a potential target foranti-proliferative therapeutics (J Neurosci Res 67(5):559-65 (2002); andProg Neurobiol 65(6):593-608 (2001)).

The mouse orphan G protein-coupled receptor FEX protein (mouse_GPCRFEX;Genbank Accession No:gi|6753842; SEQ ID NO:10) is thought to represent anovel G protein-coupled receptor expressed in follicles, that has ahigher degree of homology to glycoprotein receptors, namely those forFSH, LH, and TSH, than to other G protein-coupled receptors. FeX has 18leucine-rich repeats In the adult mouse, fex expression was detected inthe male and female gonads, the adrenal medulla, and the olfactory bulbof the brain. During embryonic development, fex transcripts weredetected transiently in various tissues, particularly in selectedregions of the central nervous system, the developing face, theintervertebral discs anlagen, and the limb buds. Based upon itsexpressed pattern, is believed to represent an important receptor forsignals controlling growth and differentiation of specific embryonictissues (Biochem. Biophys. Res. Commun. 254 (1), 273-279 (1999)).

Analysis of full-length HLLRCR-1 polypeptide indicates that it containssix leucine-rich repeat domains, and a leucine-rich repeat domain in thecarboxy terminus. HLLRCR-1 is expressed dramatically in the heart,significantly in the testis, and to a lesser extent in brain and spinalcord and may play a role in apoptosis, inflammation, and axonregeneration as related to developmental processes and diseases of theheart, the pericardium and the central nervous system.

The HLLRCR-1 polypeptide was determined to comprise a potential signalsequence from about amino acid 1 to about amino acid 67 of SEQ ID NO:2(FIGS. 1A-C) according to the SPScan computer algorithm (GeneticsComputer Group suite of programs). Based upon the predicted signalpeptide cleavage site, the mature HLLRCR-1 polypeptide is expected to befrom about amino acid 68 to about amino acid 592 of SEQ ID NO:2 (FIGS.1A-C). As this determination was based upon the prediction from acomputer algorithm, the exact physiological cleavage site may vary, asdiscussed more particularly herein. In this context, the term “about”should be construed to mean 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, or 20 more amino acids in either the N— orC-terminal direction of the above referenced polypeptide.Polynucleotides encoding these polypeptides are also provided.

In addition to the mature polypeptide above, the polynucleotidesencoding the mature HLLRCR-1 polypeptide are also encompassed by thepresent invention. Specifically, from about nucleotide position 302 toabout nucleotide position 1873 of SEQ ID NO:1 (FIGS. 1A-C).

An alternative full-length polypeptide of this gene provided as SEQ IDNO:4 (corresponding to nucleotides 1 to 517 of SEQ ID NO:2), encoded bythe polynucleotide sequence according to SEQ ID NO:3 (corresponding tonucleotides 101 to 1651 of SEQ ID NO:1), and/or encoded by thepolynucleotide contained within the deposited clone, HLLRCR-1. Thealternative form of this full-length polypeptide is referred to as“HLLRCR-1b”.

In preferred embodiments, the following HLLRCR-1 polypeptide isencompassed by the present invention: (SEQ ID NO:4)MLRLVAACPESCVVCTKDVTLCHQLTYIVAAPMTTRVLIITDGYLSSIESTNLSLLFNLALLSLSRNGIEDVQEDALHGLTMLRTLLLEHNQISSSSLTDHTFSKLHSLQVLVLSNNALRTLRGSWFRNTSGLTRLQLDGNQITNLTDSSFGGTNLHSLRYLDLSNNFISYIGKDAFRPLPQLQEVDLSRNRLAHMPDVFTPLKQLILLSLDKNQWSCTCDLHPLARFLRNYIKSSAHTLRNAKDLNCQPSTAAVAAAQSVLRLSETNCDSKAPNFTLVLKDRSPLLPGPDVALLTVLGFAGAVGLTCLGLVVFNWKLHQGKANEHTSENLCCRTFDEPLCAHEARNYHTKGYCNCHLTQENEIKVMSTVGSRKEMPLLQENSHQATSASESATLDGSFRNLKKKDRGVGSTLFCQDGRLLHSECSEPPGNMRAFNEAGLLTTYNPRKVQKLWNLEPGEVQPQTLQHHIIRTEDISSDIFRRRYATPASALAGESLEKRL TNESWQPPIEKEDNGLH.

The following encoding polynucleotide are also encompassed by thepresent invention: (SEQ ID NO:3)ATGTTGCGGTTGGTGGCAGCTTGCCCTGAGTCATGTGTGGTGTGCACCAAAGATGTAACCCTCTGTCACCAGCTAACCTATATAGTAGCAGCCCCTATGACCACGAGGGTTTTAATCATCACCGATGGATATCTCTCCTCTATTGAGAGCACAAACCTGTCTCTCTTGTTTAATCTTGCCCTGCTCTCCCTAAGCAGAAATGGTATCGAGGATGTTCAGGAAGATGCCCTGCATGGGCTTACGATGTTGCGGACCTTGTTGCTGGAGCACAACCAAATATCCAGCTCTTCGCTCACTGATCACACCTTCAGCAAGCTTCACAGCCTGCAGGTACTGGTGCTGAGCAATAATGCTCTCCGCACCCTACGAGGGTCTTGGTTCCGAAACACAAGCGGCCTGACCCGGCTCCAGCTGGATGGGAATCAGATTACTAATCTCACAGACAGTTCTTTCGGAGGCACGAATCTCCACAGTCTCAGGTATCTGGATTTATCCAACAATTTTATTTCCTACATTGGGAAAGATGCCTTCCGGCCCCTGCCTCAACTACAGGAAGTGGACCTTTCCCGAAATAGGTTAGCCCACATGCCGGATGTGTTTACTCCACTGAAGCAGTTAATCCTTCTGAGCTTAGATAAGAACCAGTGGAGCTGCACTTGTGATCTCCATCCCCTTGCTCGGTTTTTAAGAAACTACATTAAGTCTTCTGCTCACACGCTCAGGAATGCCAAGGACCTAAATTGCCAGCCATCTACCGCAGCTGTGGCAGCTGCACAGAGTGTGCTGAGGCTGTCTGAGACCAACTGTGATTCCAAAGCTCCCAACTTCACTCTGGTTCTAAAGGACAGAAGTCCCCTCCTCCCAGGACCAGATGTGGCCCTGCTGACTGTCCTTGGCTTCGCAGGAGCTGTTGGTCTCACTTGCCTAGGTTTAGTTGTATTTAACTGGAAACTCCACCAAGGCAAAGCAAATGAACACACATCAGAAAACCTTTGTTGCAGAACCTTCGATGAACCCCTGTGTGCTCATGAGGCAAGAAATTACCACACTAAGGGATACTGCAACTGCCACTTAACTCAGGAAAACGAGATAAAGGTCATGTCCACTGTGGGGTCCAGAAAAGAAATGCCACTTTTACAGGAAAATAGCCATCAAGCAACATCGGCCTCTGAGTCTGCAACCCTTGACGGATCATTTAGAAACCTGAAAAAGAAAGACCGTGGGGTAGGCAGCACTTTATTTTGCCAGGATGGTAGATTGCTGCATTCGGAATGTTCAGAGCCTCCTGGAAATATGAGAGCTTTTAATGAAGCAGGCTTACTTACAACATATAATCCAAGGAAAGTTCAAAAGCTATGGAATCTTGAGCCTGGAGAAGTCCAGCCTCAAACTCTGCAACACCATATAATAAGAACAGAAGATATCAGCAGTGACATATTTAGAAGAAGATATGCAACACCCGCTTCAGCCTTGGCAGGAGAAAGTCTTGAGAAGCGTTTAACAAATGAATCATGGCAGCCTCCAATAGAAAAAGAAGACAATGGCTTACA C.The present invention also encompasses the use of this HLLRCR-1polypeptide as an immunogenic and/or antigenic epitope as describedelsewhere herein.

The determined nucleotide sequence of the full-length HLLRCR-1b cDNA(SEQ ID NO:3) contains an open reading frame encoding a protein of about517 amino acid residues, with a deduced molecular weight of about 57.5kDa. The amino acid sequence of the predicted full-length HLLRCR-1bpolypeptide is shown is provided elsewhere herein (SEQ ID NO:4).

The HLLRCR-1b polypeptide was determined to comprise a potential signalsequence from about amino acid 1 to about amino acid 67 of SEQ ID NO:4according to the SPScan computer algorithm (Genetics Computer Groupsuite of programs). Based upon the predicted signal peptide cleavagesite, the mature full-length HLLRCR-1 polypeptide is expected to be fromabout amino acid 68 to about amino acid 517 of SEQ ID NO:4. As thisdetermination was based upon the prediction from a computer algorithm,the exact physiological cleavage site may vary, as discussed moreparticularly herein. In this context, the term “about” should beconstrued to mean 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, or 20 more amino acids in either the N— or C-terminaldirection of the above referenced polypeptide. Polynucleotides encodingthese polypeptides are also provided.

In addition to the mature polypeptide above, the polynucleotidesencoding the mature HLLRCR-1b polypeptide are also encompassed by thepresent invention. Specifically, from about nucleotide position 202 toabout nucleotide position 1551 of SEQ ID NO:4.

Based upon the strong homology to members of the leucine-rich repeatcontaining proteins, the HLLRCR-1 polypeptide is expected to share atleast some biological activity with leucine-rich repeat containingproteins, preferably with the NoGo receptor proteins, and morepreferably with leucine-rich repeat containing proteins found withincardiac tissues.

The HLLRCR-1 polypeptide was also determined to comprise severalconserved leucine residues, at amino acid 11, 217, 219, 247, 307 and/or425 of SEQ ID No: 2 (FIGS. 1A-C). The conservation of leucines at keyamino acid residues is consistent with the HLLRCR-1 polypeptide as beinga member of the leucine-rich repeat containing protein family, and maybe indicative of conserved structural features, which may correlate withconservation of protein function and/or activity.

The HLLRCR-1 polypeptide was also determined to comprise severalconserved leucines, at amino acid 4, 25, 37, 45, 55, 58, 61, 63, 87, 97,106, 108, 111, 113, 122, 132, 135, 137, 146, 155, 158, 161, 163, 179,192, 187, 192, 203, 206, 208, 210, 225, 228, 264, 277, 306, 317, 357,402, 419, 439, 451, 454, 465, 490, 495, and/or 499 of SEQ ID No: 1(FIGS. 1A-C). Conservation of leucines at key amino acid residues isindicative of conserved structural features, which may correlate withconservation of protein function and/or activity.

Expression profiling designed to measure the steady state mRNA levelsencoding the HLLRCR-1 polypeptide showed predominately high expressionlevels in heart, significant expression in testis, and to a lesserextent, in brain, spinal cord, and brain subregions (See FIG. 5).

Addition expression profiling analysis of HLRRCR-1 expression levels invarious cancer cell lines by TaqMan™ quantitative PCR (see FIG. 8)determined that HLRRCR-1 is expressed in several lung cancer cell lines,significantly in several colon cancer cell lines, and to a lesser extentin other human tumor cell lines as shown. The data suggests the HLRRCR-1polypeptide may play a critical role in the development of a transformedphenotype leading to the development of cancers and/or a proliferativecondition, either directly or indirectly. Alternatively, the HLRRCR-1polypeptide may play a protective role and could be activated inresponse to a cancerous or proliferative phenotype. Whether HLRRCR-1plays a role in directing transformation, or plays the role ofprotecting cells in response to a transformed phenotype, its role inlung and/or colon cancer tumors is likely to be enhanced relative tonormal tissues. Therefore, antagonists or agonists of the HLRRCR-1polypeptide may be useful in the treatment, amelioration, and/orprevention of a variety of proliferative conditions, including, but notlimited to lung, and colon tumors, in addition to proliferativedisorders of the lung and colon.

Expanded analysis of HLRRCR-1 expression levels by TaqMan™ quantitativePCR (see FIG. 9) confirmed that the HLRRCR-1 polypeptide is expressed inthe heart, albeit at significantly lower levels than previously observedaccording to the data shown in FIG. 5. HLRRCR-1 mRNA was expressedpredominately in the expressed predominately in hippocampus andthroughout the cortex of the brain. HLRRCR-1 was also significantexpressed in the left ventricle of the heart, the DRG, and to a lesserextent in other tissues. The low level of HLRRCR-1 expression observedin the heart was specific to the left ventricle of the heart, but at alevel that was 200 times lower than the hippocampus and cortex. HLRRCR-1expression was notably very low in the cerebellum and caudate nucleus ofthe brain. Quantitatively, HLRRCR-1 expression was highest in thehippocampus (approximately 1600 times higher than the cerebellum) anduniformly high across the seven cortex sub-regions analyzed (between 800and 1300 times higher than the cerebellum). The remaining brainsub-regions tested were shown to have have relatively low steady statelevels of HLRRCR-1. Expression of HLRRCR-1 was also found in the DRG(500 fold higher than the cerebellum). These data suggest that HLRRCR-1functions primarily in higher executive type brain functions involvingcognition, learning, the development of long term potentiation, and thusmay play an integral role, either directly or indirectly, in theestablishment and/or maintenance of memory.

HLRRCR-1 expression in the hippocampus and the fact that it containsleucine rich repeats with homology to the Nogo receptor suggests that itmay have possible involvement in the development of neurodegenerativedisorders, such as Alzheimer's and other forms of dementia, ifmis-regulated. The presence of LLR domains on HLRRCR-1, which are alsoimplicated in the process of apoptosis, suggests a possible role invarious types of epilepsy and seizures, where neuronal injury and deathin the hippocampus have clearly been shown to occur. These data suggestthat modulators of HLRRCR-1 may have utility in the treatment varioustypes of learning cognition related disorders that involve the loss ofneurons. Specifically, modulators of the HLRRCR-1 polypeptide may beuseful in the treatment, amelioration, and/or prevention of a variety ofneural conditions, including, but not limited to memory disorders,establishment of short term memory, establishment of long term memory,dimentia, cognition, learning, the development of long termpotentiation, disorders associated with neuron attrition, Alzheimers,Parkinson's, disorders that accompany sniffing of volatile vapors,disorders that accompany severe trauma to the brain, in addition toother disorders associated with the brain, particularly memorydisorders.

Moreover, an additional analysis of HLRRCR-1 expression levels byTaqMan™ quantitative PCR (see FIG. 10) in disease cells and tissuesindicated that the HLRRCR-1 polypeptide is differentially expressed inParkinson's substantia nigra tissue compared to that observed in controlsubstantia nigra RNA samples. The differential expression is significantwith a P value 0.00083 as determined by applying an ANOVA analysis tothe expression levels. In the Parkinson's substantia nigra tissueresults, an average of 3 samples were tested, and at least one of thesamples showed an approximately 250-fold induction in HLRRCR-1 steadystate RNA over that observed in 3 normal control samples. When comparedto age-matched controls the increase in steady state RNA for HLRCR-1 wasclearly not related to the age of the individual. These data support arole of HLRRCR-1 in regulating various neural disorders functions,particularly movement disorders, such as Parkinson's as well as otherforms of ataxia.

Thus, modulators of the HLRRCR-1 polypeptide may be useful in thetreatment, amelioration, and/or prevention of a variety of neuralconditions, including, but not limited to movement disorders, such asParkinson's as well as other forms of ataxia.

The HLLRCR-1 polynucleotides and polypeptides of the present invention,including agonists and/or fragments thereof, have uses that includedetecting, prognosing, treating, preventing, and/or ameliorating thefollowing diseases and/or disorders, disorders related to aberrantapoptosis regulation, disorders related to aberrant cell adhesionregulation, and disorders related to aberrant cellular proliferation,inflammation, and axon regeneration as they relate to developmentalprocesses and diseases of the heart, the pericardium and the centralnervous system, in addition to, cardiovascular, reproductive, and neuraldisorders.

The HLLRCR-1 polynucleotides and polypeptides of the present invention,including agonists and/or fragments thereof, have uses that includemodulating signal transduction activity, in various cells, tissues, andorganisms, and particularly in mammalian heart, testis, brain, andspinal cord tissue, preferably human tissue.

The strong homology to leucine-rich repeat proteins, combined with thepredominate localized expression in heart tissue suggests the HLLRCR-1polynucleotides and polypeptides may be useful in treating, diagnosing,prognosing, and/or preventing cardiovascular diseases and/or disorders,which include, but are not limited to: myocardio infarction, congestiveheart failure, arrthymias, cardiomyopathy, atherosclerosis,arterialsclerosis, microvascular disease, embolism, thromobosis,pulmonary edema, palpitation, dyspnea, angina, hypotension, syncope,heart murmer, aberrant ECG, hypertrophic cardiomyopathy, the Marfansyndrome, sudden death, prolonged QT syndrome, congenital defects,cardiac viral infections, valvular heart disease, and hypertension.

Similarly, HLLRCR-1 polynucleotides and polypeptides may be useful forameliorating cardiovascular diseases and symptoms which resultindirectly from various non-cardiavascular effects, which include, butare not limited to, the following, obesity, smoking, Down syndrome(associated with endocardial cushion defect); bony abnormalities of theupper extremities (associated with atrial septal defect in the Holt-Oramsyndrome); muscular dystrophies (associated with cardiomyopathy);hemochromatosis and glycogen storage disease (associated with myocardialinfiltration and restrictive cardiomyopathy); congenital deafness(associated with prolonged QT interval and serious cardiac arrhythmias);Raynaud's disease (associated with primary pulmonary hypertension andcoronary vasospasm); connective tissue disorders, i.e., the Marfansyndrome, Ehlers-Danlos and Hurler syndromes, and related disorders ofmucopolysaccharide metabolism (aortic dilatation, prolapsed mitralvalve, a variety of arterial abnormalities); acromegaly (hypertension,accelerated coronary atherosclerosis, conduction defects,cardiomyopathy); hyperthyroidism (heart failure, atrial fibrillation);hypothyroidism (pericardial effusion, coronary artery disease);rheumatoid arthritis (pericarditis, aortic valve disease); scleroderma(cor pulmonale, myocardial fibrosis, pericarditis); systemic lupuserythematosus (valvulitis, myocarditis, pericarditis); sarcoidosis(arrhythmias, cardiomyopathy); postmenopausal effects, Chlamydialinfections, polycystic ovary disease, thyroid disease, alcoholism, diet,and exfoliative dermatitis (high-output heart failure), for example.

Moreover, polynucleotides and polypeptides, including fragments and/orantagonists thereof, have uses which include, directly or indirectly,treating, preventing, diagnosing, and/or prognosing the following,non-limiting, cardiovascular infections: blood stream invasion,bacteremia, sepsis, Streptococcus pneumoniae infection, group astreptococci infection, group b streptococci infection, Enterococcusinfection, nonenterococcal group D streptococci infection,nonenterococcal group C streptococci infection, nonenterococcal group Gstreptococci infection, Streptoccus viridans infection, Staphylococcusaureus infection, coagulase-negative staphylococci infection,gram-negative Bacilli infection, Enterobacteriaceae infection,Psudomonas spp. Infection, Acinobacter spp. Infection, Flavobacteriummeningosepticum infection, Aeromonas spp. Infection, Stenotrophomonasmaltophilia infection, gram-negative coccobacilli infection, Haemophilusinfluenza infection, Branhamella catarrhalis infection, anaerobeinfection, Bacteriodes fragilis infection, Clostridium infection, fungalinfection, Candida spp. Infection, non-albicans Candida spp. Infection,Hansenula anomala infection, Malassezia furfur infection, nontuberculousMycobacteria infection, Mycobacterium avium infection, Mycobacteriumchelonae infection, Mycobacterium fortuitum infection, spirochetalinfection, Borrelia burgdorferi infection, in addition to any othercardiovascular disease and/or disorder (e.g., non-sepsis) implicated bythe causative agents listed above or elsewhere herein.

Several leucine-rich repeat proteins have been identified that areexpressed in heart tissue. Non-limiting examples include the Asporin(Lorenzo, P., Aspberg, A., Onnerfjord, P., Bayliss, M, T., Neame, P, J.,Heinegard, D, J. Biol, Chem., 276(15):12201-11, (2001)), LRRFIP1 andLRRFIP2 (Fong, K, S., de, Couet, H, G, Genomics., 58(2):146-57, (1999)),FLRT2, and FLRT3 (Lacy, S, E., Bonnemann, C, G., Buzney, E, A., Kunkel,L, M, Genomics., 62(3):417-26, (1999)), ISLR (Nagasawa, A., Kubota, R.,Imamura, Y., Nagamine, K., Wang, Y., Asakawa, S., Kudoh, J., Minoshima,S., Mashima, Y., Oguchi, Y., Shimizu, N, Genomics., 44(3):273-9,(1997)), and RP105 (Miyake, K., Yamashita, Y., Ogata, M., Sudo, T.,Kimoto, M, J. Immunol., 154(7):3333-40, (1995)).

The strong homology to leucine-rich repeat proteins, combined with thelocalized expression in testis tissue suggests the potential utility forHLLRCR-1 polynucleotides and polypeptides in treating, diagnosing,prognosing, and/or preventing testicular, in addition to reproductivedisorders.

In preferred embodiments, HLLRCR-1 polynucleotides and polypeptidesincluding agonists and fragments thereof, have uses which includetreating, diagnosing, prognosing, and/or preventing the following,non-limiting, diseases or disorders of the testis: spermatogenesis,infertility, Klinefelter's syndrome, XX male, epididymitis, genitalwarts, germinal cell aplasia, cryptorchidism, varicocele, immotile ciliasyndrome, and viral orchitis. The HLLRCR-1 polynucleotides andpolypeptides including agonists and fragments thereof, may also haveuses related to modulating testicular development, embryogenesis,reproduction, and in ameliorating, treating, and/or preventingtesticular proliferative disorders (e.g., cancers, which include, forexample, choriocarcinoma, Nonseminoma, seminona, and testicular germcell tumors).

Likewise, the localized expression in testis tissue also emphasizes thepotential utility for HLLRCR-1 polynucleotides and polypeptides intreating, diagnosing, prognosing, and/or preventing metabolic diseasesand disorders which include the following, not limiting examples:premature puberty, incomplete puberty, Kallman syndrome, Cushing'ssyndrome, hyperprolactinemia, hemochromatosis, congenital adrenalhyperplasia, FSH deficiency, and granulomatous disease, for example.

This gene product may also be useful in assays designed to identifybinding agents, as such agents (antagonists) are useful as malecontraceptive agents. The testes are also a site of active geneexpression of transcripts that is expressed, particularly at low levels,in other tissues of the body. Therefore, this gene product may beexpressed in other specific tissues or organs where it may play relatedfunctional roles in other processes, such as hematopoiesis,inflammation, bone formation, and kidney function, to name a fewpossible target indications.

The HLLRCR-1 polynucleotides and polypeptides, including agonists,antagonists, and/or fragments thereof, of the present invention haveuses which include, for example, modulating cellular proliferation.Likewise, the HLLRCR-1 polynucleotides and polypeptides, includingagonists, antagonists, and/or fragments thereof, may be useful for thetreatment, detection, amelioration, and/or prevention of disordersrelated, or directly linked to, aberrant cellular proliferation, suchas, for example, cancers.

Moreover, HLLRCR-1 polynucleotides and polypeptides, including fragmentsand agonists thereof, may have uses which include treating, diagnosing,prognosing, and/or preventing hyperproliferative disorders, particularlyof the cardiovascular, reproductive, and nervous systems. Such disordersmay include, for example, cancers, and metastasis.

The HLLRCR-1 polynucleotides and polypeptides, including fragments andagonists thereof, may have uses which include, either directly orindirectly, for boosting immune responses.

The HLLRCR-1 polynucleotides and polypeptides, including fragmentsand/or antagonsists thereof, may have uses which include identificationof modulators of HLLRCR-1 function including antibodies (for detectionor neutralization), naturally-occurring modulators and small moleculemodulators. Antibodies to domains of the HLLRCR-1 protein could be usedas diagnostic agents of hematopoietic and inflammatory conditions inpatients, are useful in monitoring the activation of signal transductionpathways, and can be used as a biomarker for the involvement ofleucine-rich repeat containing proteins in disease states, and in theevaluation of inhibitors of leucine-rich repeat containing proteins invivo.

HLLRCR-1 polypeptides and polynucleotides have additional uses whichinclude diagnosing diseases related to the over and/or under expressionof HLLRCR-1 by identifying mutations in the HLLRCR-1 gene by usingHLLRCR-1 sequences as probes or by determining HLLRCR-1 protein or mRNAexpression levels. HLLRCR-1 polypeptides may be useful forscreening-compounds that affect the activity of the protein. HLLRCR-1peptides can also be used for the generation of specific antibodies andas bait in yeast two hybrid screens to find proteins the specificallyinteract with HLLRCR-1 (described elsewhere herein).

Recently, leucine-rich repeat containing proteins have been directlyimplicated in the pathogenesis of Bernard-Soulier syndrome (BSS), ahereditary qualitative platelet disorder. The disorder has been linkedto the qualitative or quantitative abnormality of the plateletglycoprotein (GP) Ib/IX/V complex, which is formed by the aggregation ofseveral leucine-rich repeat containing proteins (Hayashi, T.; Suzuki,K., Semin. Thromb. Hemost.,26(1): 53-9 (2000).

In preferred embodiments, HLLRCR-1 polypeptides, including antagonists,and fragments thereof, have uses which include, for example, thetreatment, detection, prevention, prognosis, and/or amelioration ofplatelet disorders, including, but not limited to, Bernard-Souliersyndrome (BSS).

As descussed elsewhere herein, the Drosophia Toll proteins, includingthe human homologues thereof, have been implicated in modulatingdevelopment and in non-infectious disease (Schuster, J M., Nelson, P S,J. Leukoc, Biol., 67(6):767-73, (2000)).

In preferred embodiments, HLLRCR-1 polypeptides, including antagonists,and fragments thereof, have uses which include, for example, thetreatment, detection, prevention, prognosis, and/or amelioration ofdevelopmental disorders, and non-infectious disorders, such as innateimmunity to bacterial pathogens, adaptive immune responses, and othersas listed in Schuster, J M et al., supra.

Leucine-rich repeat proteins have also been implicated in the incidenceof systemic lupus erythematosus (Koarada, S., Tada, Y., Ushiyama, O.,Morito, F., Suzuki, N., Ohta, A., Miyake, K., Kimoto, M., Nagasawa, K,Arthritis, Rheum., 42(12):2593-600, (1999)).

In preferred embodiments, HLLRCR-1 polypeptides, including antagonists,and fragments thereof, have uses which include, for example, thetreatment, detection, prevention, prognosis, and/or amelioration ofimmune disorders, which include, for example, systemic lupuserythematosus.

As described elsewhere herein, leucine-rich repeat proteins are alsoimplicated in a number of processes related to pathogen and/or diseaseresistance in plants. This is of particular significance, since mammals,including, humans, have homologues of some of these proteins which arethought to function in innate immune responses ((Dixon, M S.,Hatzixanthis, K., Jones, D A., Harrison, K., Jones, J D, Plant, Cell.,10(11):1915-25, (1998); Ellis, J., Jones, D, Curr, Opin, Plant, Biol.,1(4):288-93, (1998); Collins, N., Drake, J., Ayliffe, M., Sun, Q.,Ellis, J., Hulbert, S., Pryor, T, Plant, Cell., 11(7):1365-76, (1999);Wang, Z X., Yano, M., Yamanouchi, U., Iwamoto, M., Monna, L., Hayasaka,H., Katayose, Y., Sasaki, T, Plant, J., 19(1):55-64, (1999); Richter, TE., Ronald, P C, Plant, Mol, Biol., 42(1):195-204, (2000); Graham, M A.,k, L F., Lohnes, D., Cregan, P., Shoemaker, R C, Genome., 43(1):86-93,(2000); and He, Z., Wang, Z Y., Li, J., Zhu, Q., Lamb, C., Ronald, P.,Chory, J. Science. 30., 288(5475):2360-3, (2000)).

Although it is believed the encoded polypeptide may share at least somebiological activities with leucine-rich repeat containing proteins(particularly Nogo receptor proteins), a number of methods ofdetermining the exact biological function of this clone are either knownin the art or are described elsewhere herein. Briefly, the function ofthis clone may be determined by applying microarray methodology. Nucleicacids corresponding to the HLLRCR-1 polynucleotides, in addition to,other clones of the present invention, may be arrayed on microchips forexpression profiling. Depending on which polynucleotide probe is used tohybridize to the slides, a change in expression of a specific gene mayprovide additional insight into the function of this gene based upon theconditions being studied. For example, an observed increase or decreasein expression levels when the polynucleotide probe used comes fromdiseased heart tissue, as compared to, normal tissue might indicate afunction in modulating heart function, for example. In the case ofHLLRCR-1, heart, testis, brain, and/or spinal cord tissue should beused, for example, to extract RNA to prepare the probe.

In addition, the function of the protein may be assessed by applyingquantitative PCR methodology, for example. Real time quantitative PCRwould provide the capability of following the expression of the HLLRCR-1gene throughout development, for example. Quantitative PCR methodologyrequires only a nominal amount of tissue from each developmentallyimportant step is needed to perform such experiments Therefore, theapplication of quantitative PCR methodology to refining the biologicalfunction of this polypeptide is encompassed by the present invention. Inthe case of HLLRCR-1, a disease correlation related to HLLRCR-1 may bemade by comparing the mRNA expression level of HLLRCR-1 in normaltissue, as compared to diseased tissue (particularly diseased tissueisolated from the following: heart, testis, brain, and/or spinal cordtissue). Significantly higher or lower levels of HLLRCR-1 expression inthe diseased tissue may suggest HLLRCR-1 plays a role in diseaseprogression, and antagonists against HLLRCR-1 polypeptides would beuseful therapeutically in treating, preventing, and/or ameliorating thedisease. Alternatively, significantly higher or lower levels of HLLRCR-1expression in the diseased tissue may suggest HLLRCR-1 plays a defensiverole against disease progression, and agonists of HLLRCR-1 polypeptidesmay be useful therapeutically in treating, preventing, and/orameliorating the disease. Also encompassed by the present invention arequantitative PCR probes corresponding to the polynucleotide sequenceprovided as SEQ ID NO:1 (FIGS. 1A-C).

The function of the protein may also be assessed through complementationassays in yeast. For example, in the case of the HLLRCR-1, transformingyeast deficient in leucine-rich repeat containing protein activity,preferably NoGo receptor activity, for example, and assessing theirability to grow would provide convincing evidence the HLLRCR-1polypeptide has leucine-rich repeat containing protein activity, andpotentially NoGo receptor activity. Additional assay conditions andmethods that may be used in assessing the function of thepolynucleotides and polypeptides of the present invention are known inthe art, some of which are disclosed elsewhere herein.

Alternatively, the biological function of the encoded polypeptide may bedetermined by disrupting a homologue of this polypeptide in Mice and/orrats and observing the resulting phenotype. Such knock-out experimentsare known in the art, some of which are disclosed elsewhere herein.

Moreover, the biological function of this polypeptide may be determinedby the application of antisense and/or sense methodology and theresulting generation of transgenic mice and/or rats. Expressing aparticular gene in either sense or antisense orientation in a transgenicmouse or rat could lead to respectively higher or lower expressionlevels of that particular gene. Altering the endogenous expressionlevels of a gene can lead to the observation of a particular phenotypethat can then be used to derive indications on the function of the gene.The gene can be either over-expressed or under expressed in every cellof the organism at all times using a strong ubiquitous promoter, or itcould be expressed in one or more discrete parts of the organism using awell characterized tissue-specific promoter (e.g., a heart, testis,brain, and/or spinal cord-specific promoter), or it can be expressed ata specified time of development using an inducible and/or adevelopmentally regulated promoter.

In the case of HLLRCR-1 transgenic mice or rats, if no phenotype isapparent in normal growth conditions, observing the organism underdiseased conditions (cardiovascular, reproductive, and/or neuraldisorders, in addition to cancers, etc.) may lead to understanding thefunction of the gene. Therefore, the application of antisense and/orsense methodology to the creation of transgenic mice or rats to refinethe biological function of the polypeptide is encompassed by the presentinvention.

In preferred embodiments, the following N-terminal HLRRCR-1 deletionpolypeptides are encompassed by the present invention: M1-K591, L2-K591,R3-K591, L4-K591, V5-K591, A6-K591, A7-K591, C8-K591, P9-K591, E10-K591,S11-K591, C12-K591, V13-K591, V14-K591, C15-K591, T16-K591, K17-K591,D18-K591, V19-K591, T20-K591, L21-K591, C22-K591, H23-K591, Q24-K591,L25-K591, T26-K591, Y27-K591, 128-K591, V29-K591, A30-K591, A31-K591,P32-K591, M33-K591, T34-K591, T35-K591, R36-K591, V37-K591, L38-K591,I39-K591, I40-K591, T41-K591, D42-K591, G43-K591, Y44-K591, L45-K591,S46-K591, S47-K591, I48-K591, E49-K591, S50-K591, T51-K591, N52-K591,L53-K591, S54-K591, L55-K591, L56-K591, F57-K591, N58-K591, L59-K591,A60-K591, L61-K591, L62-K591, S63-K591, L64-K591, S65-K591, R66-K591,N67-K591, G68-K591, I69-K591, E70-K591, D71-K591, V72-K591, Q73-K591,E74-K591, D75-K591, A76-K591, L77-K591, H78-K591, G79-K591, L80-K591,T81-K591, M82-K591, L83-K591, R84-K591, T85-K591, L86-K591, L87-K591,L88-K591, E89-K591, H90-K591, N91-K591, Q92-K591, 193-K591, S94-K591,S95-K591, S96-K591, S97-K591, L98-K591, T99-K591, D100-K591, H101-K591,T102-K591, F103-K591, S104-K591, K105-K591, L106-K591, H107-K591,S108-K591, L109-K591, Q110-K591, V111-K591, L112-K591, V113-K591,L114-K591, S115-K591, N116-K591, N117-K591, A118-K591, L119-K591,R120-K591, T121-K591, L122-K591, R123-K591, G124-K591, S125-K591,W126-K591, F127-K591, R128-K591, N129-K591, T130-K591, S131-K591,G132-K591, L133-K591, T134-K591, R135-K591, L136-K591, Q137-K591,L138-K591, D139-K591, G140-K591, N141-K591, Q142-K591, I143-K591,T144-K591, N145-K591, L146-K591, T147-K591, D148-K591, S149-K591,S150-K591, F151-K591, G152-K591, G153-K591, T154-K591, N155-K591,L156-K591, H157-K591, S158-K591, L159-K591, R160-K591, Y161-K591,L162-K591, D163-K591, L164-K591, S165-K591, N166-K591, N167-K591,F168-K591, I169-K591, S170-K591, Y171-K591, I172-K591, G173-K591,K174-K591, D175-K591, A176-K591, F177-K591, R178-K591, P179-K591,L180-K591, P181-K591, Q182-K591, L183-K591, Q184-K591, E185-K591,V186-K591, D187-K591, L188-K591, S189-K591, R190-K591, N191-K591,R192-K591, L193-K591, A194-K591, H195-K591, M196-K591, P197-K591,D198-K591, V199-K591-F200-K591, T201-K591, P202-K591, L203-K591,K204-K591, Q205-K591, L206-K591, I207-K591, L208-K591, L209-K591,S210-K591, L211-K591, D212-K591, K213-K591, N214-K591, Q215-K591,W216-K591, S217-K591, C218-K591, T219-K591, C220-K591, D221-K591,L222-K591, H223-K591, P224-K591, L225-K591, A226-K591, R227-K591,F228-K591, L220-K591, R230-K591, N231-K591, Y232-K591, I233-K591,K234-K591, S235-K591, S236-K591, A237-K591, H238-K591, T239-K591,L240-K591, R241-K591, N242-K591, A243-K591, K244-K591, D245-K591,L246-K591, N247-K591, C248-K591, Q249-K591, P250-K591, S251-K581,T252-K591, A253-K591, A254-K591, V255-K591, A256-K591, A257-K591,A258-K591, Q259-K591, S260-K591, V261-K591, L262-K591, R263-K591,L264-K591, S265-K591, E266-K591, T267-K591, N268-K591, C269-K591,D270-K591, S271-K591, K272-K591, A273-K591, P274-K591, N275-K591,F276-K591, T277-K591, L278-K591, V279-K591, L280-K591, K281-K591,D282-K591, R283-K591, S284-K591, P285-K591, L286-K591, L287-K591,P288-K591, G289-K591, P290-K591, D291-K591, V292-K591, A293-K591,L294-K591, L295-K591, T296-K591, V297-K591, L298-K591, G299-K591,F300-K591, A301-K591, G302-K591, A303-K591, V304-K591, G305-K591,L306-K591, T307-K591, C308-K591, L309-K591, G310-K591, L311-K591,V312-K591, V313-K591, F314-K591, N315-K591, W316-K591, K317-K591,L318-K591, H319-K591, Q320-K591, G321-K591, K322-K591, A332-K591,N324-K591, E325-K591, H326-K591, T327-K591, S328-K591, E329-K591,N330-K591, L331-K591, C332-K591, C333-K591, R334-K591, T335-K591,F336-K591, D337-K591, E338-K591, P339-K591, L340-K591, C341-K591,A342-K591, H343-K591, E344-K591, A345-K591, R346-K591, N347-K591,Y348-K591, H349-K591, T350-K591, K351-K591, G352-K591, Y353-K591,C354-K591, N355-K591, C356-K591, H357-K591, L358-K591, T359-K591,Q360-K591, E361-K591, N362-K591, E363-K591, I364-K591, K365-K591,V366-K591, M367-K591, S368-K591, T369-K591, V370-K591, G371-K591,S372-K591, R373-K591, K374-K591, E375-K591, M376-K591, P377-K591,L378-K591, L379-K591, Q380-K591, E381-K591, N382-K591, S383-K591,H384-K591, Q385-K591, A386-K591, T387-K591, S388-K591, A389-K591,S390-K591, E391-K591, S392-K591, A393-K591, T394-K591, L395-K591,D396-K591, G397-K591, S398-K591, F399-K591, R400-K591, N401-K591,L402-K591, K403-K591, K404-K591, K405-K591, D406-K591, R407-K591,G408-K591, V409-K591, G410-K591, S411-K591, T412-K591, L413-K591,F414-K591, C415-K591, Q416-K591, D417-K591, G418-K591, R419-K591,L420-K591, L421-K591, H422-K591, S423-K591, E424-K591, C425-K591,S426-K591, E427-K591, P428-K591, P429-K591; G430-K591, N431-K591,M432-K591, R433-K591, A434-K591, F435-K591, N436-K591, E437-K591,A438-K591, G439-K591, L440-K591, L441-K591, T442-K591, T443-K591,Y444-K591, N445-K591, P446-K591, R447-K591, K448-K591, V449-K591,Q450-K591, K451-K591, L452-K591, W453-K591, N454-K591, L455-K591,E456-K591, P457-K591, G458-K591, E459-K591, V460-K591, Q461-K591,P462-K591, Q463-K591, T464-K591, L465-K591, Q466-K591, H467-K591,H468-K591, I469-K591, I470-K591, R471-K591, T472-K591, E473-K591,D474-K591, I475-K591, S476-K591, S477-K591, D478-K591, I479-K591,F480-K591, R481-K591, R482-K591, R483-K591, Y484-K591, A485-K591,T486-K591, P487-K591, A488-K591, S489-K591, A490-K591, L491-K591,A492-K591, G493-K591, E494-K591, S495-K591, L496-K591, E497-K591,K498-K591, R499-K591, L500-K591, T501-K591, N502-K591, E503-K591,S504-K591, W505-K591, Q506-K591, P507-K591, P508-K591, 1509-K591,E510-K591, K511-K591, E512-K591, D513-K591, N514-K591, G515-K591,L516-K591, H517-K591, P518-K591, H519-K591, R520-K591, Q521-K591,R522-K591, H523-K591, F524-K591, I525-K591, T526-K591, S527-K591,S528-K591, S529-K591, S530-K591, K531-K591, P532-K591, C533-K591,E534-K591, P535-K591, E536-K591, E537-K591, H538-K591, Y539-K591,V540-K591, Q541-K591, K542-K591, I543-K591, V544-K591, Q545-K591,K546-K591, N547-K591, R548-K591, S549-K591, K550-K591, Y551-K591,D552-K591, D553-K591, P554-K591, C555-K591, G556-K591, L557-K591,L558-K591, K559-K591, Q560-K591, S561-K591, K562-K591, P563-K591,R564-K591, Y565-K591, F566-K591, Q567-K591, P568-K591, N569-K591,N570-K591, S571-K591, L572-K591, I573-K591, C574-K591, K575-K591,Y576-K591, V577-K591, P578-K591, C579-K591, E580-K591, Q581-K591,F582-K591, E583-K591, D584-K591, and/or Y585-K591 of SEQ ID NO:2.Polynucleotide sequences encoding these polypeptides are also provided.The present invention also encompasses the use of these N-terminalHLRRCR-1 deletion polypeptides as immunogenic and/or antigenic epitopesas described elsewhere herein.

In preferred embodiments, the following C-terminal HLRRCR-1 deletionpolypeptides are encompassed by the present invention: M1-K591, M1-K590,M1-K589, M1-K588, M1-K587, M1-M586, M1-Y585, M1-D584, M1-E583, M1-F582,M1-Q581, M1-E580, M1-C579, M1-P578, M1-V577, M1-Y576, M1-K575, M1-C574,M1-I573, M1-L572, M1-S571, M1-N570, M1-N569, M1-P568, M1-Q567, M1-F566,M1-Y565, M1-R564, M1-P563, M1-K562, M1-S561, M1-Q560, M1-K559, M1-L558,M1-L557, M1-G556, M1-C555, M1-P554, M1-D553, M1-D552, M1-Y551, M1-K550,M1-S549, M1-R548, M1-N547, M1-K546, M1-Q545, M1-V544, M1-1543, M1-K542,M1-Q541, M1-V540, M1-Y539, M1-H538, M1-E537, M1-E536, M1-P535, M1-E534,M1-C533, M1-P532, M1-K531, M1-S530, M1-S529, M1-S528, M1-S527, M1-T526,M1-I525, M1-F524, M1-H523, M1-R522, M1-Q521, M1-R520, M1-H519, M1-P518,M1-H517, M1-L516, M1-G515, M1-N514, M1-D513, M1-E512, M1-K511, M1-E510,M1-I509, M1-P508, M1-P507, M1-Q506, M1-W505, M1-S504, M1-E503, M1-N502,M1-T501, M1-L500, M1-R499, M1-K498, M1-E497, M1-L496, M1-S495, M1-E494,M1-G493, M1-A492, M1-L491, M1-A490, M1-S489, M1-A488, M1-P487, M1-T486,M1-A485, M1-Y484, M1-R483, M1-R482, M1-R481, M1-F480, M1-I479, M1-D478,M1-S477, M1-S476, M1-I475, M1-D474, M1-E473, M1-T472, M1-R471, M1-I470,M1-I469, M1-H467, M1-Q466, M1-L465, M1-T464, M1-Q463, M1-P462, M1-Q461,M1-V460, M1-E459, M1-G458, M1-P457, M1-E456, M1-L455, M1-N454, M1-W453,M1-L452, M1-K451, M1-Q450, M1-V449, M1-K448, M1-R447, M1-P446, M1-N445,M1-Y444, M1-T443, M1-T442, M1-L441, M1-L440, M1-G439, M1-A438, M1E437,M1-N436, M1-F435, M1-A434, M1-R433, M1-M432, M1-N431, M1-G430, M1-P429,M1-P428, M1-E427, M1-S426, M1-C425, M1-E424, M1-S423, M1-H422, M1-L421,M1-L420, M1-R419, M1-G418, M1-D417, M1-Q416, M1-C415, M1-F414, M1-L413,M1-T412, M1-S411, M1-G410, M1-V409, M1-G408, M1-R407, M1-D406, M1-K405,M1-K404, M1-K403, M1-L402, M1-N401, M1-R400, M1-F399, M1-S398, M1-G397,M1-D396, M1-L395, M1-T394, M1-A393, M1-S392, M1-E391, M1-S390, M1-A389,M1-S388, M1-T387, M1-A386, M1-Q385, M1-H384, M1-S383, M1-N382, M1-E381,M1-Q380, M1-L379, M1-L378, M1-P377, M1-M376, M1-E375, M1-K374, M1-R373,M1-S372, M1-G371, M1-V370, M1-T369, M1-S368, M1-M367, M1-V366, M1-K365,M1-I364, M1-E363, M1-N362, M1-E361, M1-Q360, M1-T359, M1-L358, M1-H357,M1-C356, M1-N355, M1-C354, M1-Y353, M1-G352, M1-K351, M1-T350, M1-H349,M1-Y348, M1-N347, M1-R346, M1-A345, M1-E344, M1-H343, M1-A342, M1-C341,M1-L340, M1-P339, M1-E338, M1-D337, M1-F336, M1-T335, M1-R334, M1-C333,M1-C332, M1-L331, M1-N330, M1-E329, M1-S328, M1-T327, M1-H326, M1-E325,M1-N324, M1-A323, M1-K322, M1-G321, M1-Q320, M1-H319, M1-L318, M1-K317,M1-K317, M1-W316, M1-N315, M1-F314, M1-V313, M1-V312, M1-L311, M1-G310,M1-L309, M1-C308, M1-T307, M1-L306, M1-G305, M1-V304, M1-A303, M1-G302,M1-A301, M1-F300, M1-G299, M1-L298, M1-V297, M1-T296, M1-L295, M1-L294,M1-A293, M1-V292, M1-D291, M1-P290, M1-G289, M1-P288, M1-L287, M1-L286,M1-P285, M1-S284, M1-R283, M1-D282, M1-K281, M1-L280, M1-V279, M1-L278,M1-T277, M1-F276, M1-N275, M1-P274, M1-A273, M1-K272, M1-S271, M1-D270,M1-C269, M1-N268, M1-T267, M1-E266, M1-S265, M1-L264, M1-R263, M1-L262,M1-V261, M1-S260, M1-Q259, M1-A258, M1-A257, M1-A256, M1-V255, M1-A254,M1-A253, M1-T252, M1-S251, M1-P250, M1-Q249, M1-C248, M1-N247, M1-L246,M1-D245, M1-K244, M1-A243, M1-N242, M1-R241, M1-L240, M1-T239, M1-H238,M1-A237, M1-S236, M1-S235, M1-K234, M1-I233, M1-Y232, M1-N231, M1-R230,M1-L229, M1-F228, M1-R227, M1-A226, M1-L225, M1-P224, M1-H223, M1-L222,M1-D221, M1-C220, M1-T219, M1-C218, M1-S217, M1-W216, M1-Q215, M1-N214,M1-K213, M1-D212, M1-L211, M1-S210, M1-L209, M1-L208, M1-1207, M1-L206,M1-Q205, M1-K204, M1-L203, M1-P202, M1-T201, M1-F200, M1-V199, M1-D198,M1-P197, M1-M196, M1-H195, M1-A194, M1-L193, M1-R192, M1-N191, M1-R190,M1-S189, M1-L188, M1-D187, M1-V186, M1-E185, M1-Q184, M1-L183, M1-Q182,M1-P181, M1-L180, M1-P179, M1-R178, M1-F177, M1-A176, M1-D175, M1-K174,M1-G173, M1-I172, M1-Y171, M1-S170, M1-1169, M1-F168, M1-N167, M1-N166,M1-S165, M1-L164, M1-D163, M1-L162, M1-Y161, M1-R160, M1-L159, M1-S158,M1-H157, M1-L156, M1-N155, M1-T154, M1-G153, M1-G152, M1-F151, M1-S150,M1-S149, M1-D148, M1-T147, M1-L146, M1-N145, M1-T144, M1-I143, M1-Q142,M1-N141, M1-G140, M1-D139, M1-L138, M1-Q137, M1-L136, M1-R135, M1-T134,M1-L133, M1-G132, M1-S131, M1-T130, M1-N129, M1-R128, M1-F127, M1-W126,M1-S125, M1-G124, M1-R123, M1-L122, M1-T121, M1-R120, M1-L119, M1-A118,M1-N117, M1-N116, M1-S115, M1-L114, M1-V113, M1-L112, M1-V111, M1-Q110,M1-L109, M1-S108, M1-H107, M1-L106, M1-K105, M1-S104, M1-F103, M1-T102,M1-H101, M1-D100, M1-T99, M1-L98, M1-S97, M1-S96, M1-S95, M1-S94,M1-I93, M1-Q92, M1-N91, M1-H90, M1-E89, M1-L88, M1-L87, M1-L86, M1-T85,M1-R84, M1-L83, M1-M82, M1-T81; M1-L80, M1-G79, M1-H78, M1-L77, M1-A76,M1-D75, M1-E74, M1-Q73, M1-V72, M1-D71, M1-E70, M1-I69, M1-G68, M1-N67,M1-R66, M1-S65, M1-L64, M1-S63, M1-L62, M1-L61, M1-A60, M1-L59, M1-N58,M1-F57, M1-L56, M1-L55, M1-S54, M1-L53, M52, M1-T51, M1-S50, M1-E49,M1-I48, M1-S47, M1-S46, M1-L45, M1-Y44, M1-G43, M1-D42, M1-T41, M1-I40,M1-I39, M1-L38, M1-V37, M1-R36, M1-T35, M1-T34, M1-M33, M1-P32, M1-A31,M1-A30, M1-V29, M1-I28, M1-Y27, M1-T26, M1-L25, M1-Q24, M1-H23, M1-C22,M1-L21, M1-T20, M1-V19, M1-D18, M1-K17, M1-T16, M1-C15, M1-V14, M1-V13,M1-C12, M1-S11, M1-E10, M1-P9, M1-C8, and/or M1-A7 of SEQ ID NO:2.Polynucleotide sequences encoding these polypeptides are also provided.The present invention also encompasses the use of these C-terminalHLRRCR-1 deletion polypeptides as immunogenic and/or antigenic epitopesas described elsewhere herein.

Alternatively, preferred polypeptides of the present invention maycomprise polypeptide sequences corresponding to, for example, internalregions of the HLRRCR-1 polypeptide (e.g., any combination of both N—and C-terminal HLRRCR-1 polypeptide deletions) of SEQ ID NO:2. Forexample, internal regions could be defined by the equation: amino acidNX to amino acid CX, wherein NX refers to any N-terminal deletionpolypeptide amino acid of HLRRCR-1 (SEQ ID NO:2), and where CX refers toany C-terminal deletion polypeptide amino acid of HLRRCR-1 (SEQ IDNO:2). Polynucleotides encoding these polypeptides are also provided.The present invention also encompasses the use of these polypeptides asan immunogenic and/or antigenic epitope as described elsewhere herein.

The present invention also encompasses immunogenic and/or antigenicepitopes of the HLLRCR-1 polypeptide.

The HLLRCR-1 polypeptides of the present invention were determined tocomprise several phosphorylation sites based upon the Motif algorithm(Genetics Computer Group, Inc.). The phosphorylation of such sites mayregulate some biological activity of the HLLRCR-1 polypeptide. Forexample, phosphorylation at specific sites may be involved in regulatingthe proteins ability to associate or bind to other molecules (e.g.,proteins, ligands, substrates, DNA, etc.). In the present case,phosphorylation may modulate the ability of the HLLRCR-1 polypeptide toassociate with other polypeptides, particularly cognate ligand for theHLLRCR-1 polypeptide, or its ability to modulate certain cellular signalpathways.

The HLLRCR-1 polypeptide was predicted to comprise six PKCphosphorylation sites using the Motif algorithm (Genetics ComputerGroup, Inc.). In vivo, protein kinase C exhibits a preference for thephosphorylation of serine or threonine residues. The PKC phosphorylationsites have the following consensus pattern: [ST]-x-[RK], where S or Trepresents the site of phosphorylation and ‘x’ an intervening amino acidresidue. Additional information regarding PKC phosphorylation sites canbe found in Woodget J. R., Gould K. L., Hunter T., Eur. J. Biochem.161:177-184(1986), and Kishimoto A., Nishiyama K., Nakanishi H.,Uratsuji Y., Nomura H., Takeyama Y., Nishizuka Y., J. Biol. Chem.260:12492-12499(1985); which are hereby incorporated by referenceherein.

In preferred embodiments, the following HLLRCR-1 PKC phosphorylationsite polypeptides are encompassed by the present invention:VAAPMTTRVLIIT (SEQ ID NO:22), NNALRTLRGSWFR (SEQ ID NO:23),GTNLHSLRYLDLS (SEQ ID NO:24), KSSAHTLRNAKDL (SEQ ID NO:25),MSTVGSRKEMPLL (SEQ ID NO:26), and/or ATLDGSFRNLKKK (SEQ ID NO:27).Polynucleotides encoding these polypeptides are also provided. Thepresent invention also encompasses the use of these HLLRCR-1 PKCphosphorylation site polypeptides as immunogenic and/or antigenicepitopes as described elsewhere herein.

The HLLRCR-1 polypeptide was predicted to comprise one cAMP- andcGMP-dependent protein kinase phosphorylation site using the Motifalgorithm (Genetics Computer Group, Inc.). There has been a number ofstudies relative to the specificity of cAMP- and cGMP-dependent proteinkinases. Both types of kinases appear to share a preference for thephosphorylation of serine or threonine residues found close to at leasttwo consecutive N-terminal basic residues.

A consensus pattern for cAMP- and cGMP-dependent protein kinasephosphorylation sites is as follows: [RK](2)-x-[ST], wherein “x”represents any amino acid, and S or T is the phosphorylation site.

Additional information specific to cAMP- and cGMP-dependent proteinkinase phosphorylation sites may be found in reference to the followingpublication: Fremisco J. R., Glass D. B., Krebs E. G, J. Biol. Chem.255:4240-4245(1980); Glass D. B., Smith S. B., J. Biol. Chem.258:14797-14803(1983); and Glass D. B., E1-Maghrabi M. R., Pilkis S. J.,J. Biol. Chem. 261:2987-2993(1986); which is hereby incorporated hereinin its entirety.

In preferred embodiments, the following cAMP- and cGMP-dependent proteinkinase phosphorylation site polypeptide is encompassed by the presentinvention: GESLEKRLTNESWQ (SEQ ID NO:28). Polynucleotides encoding thispolypeptide are also provided. The present invention also encompassesthe use of this cAMP- and cGMP-dependent protein kinase phosphorylationsite polypeptide as an immunogenic and/or antigenic epitope as describedelsewhere herein.

The HLLRCR-1 polypeptide was predicted to comprise six casein kinase IIphosphorylation sites using the Motif algorithm (Genetics ComputerGroup, Inc.). Casein kinase II (CK-2) is a protein serine/threoninekinase whose activity is independent of cyclic nucleotides and calcium.CK-2 phosphorylates many different proteins. The substrate specificity[1] of this enzyme can be summarized as follows: (1) Under comparableconditions Ser is favored over Thr.; (2) An acidic residue (either Aspor Glu) must be present three residues from the C-terminal of thephosphate acceptor site; (3) Additional acidic residues in positions +1,+2, +4, and +5 increase the phosphorylation rate. Most physiologicalsubstrates have at least one acidic residue in these positions; (4) Aspis preferred to Glu as the provider of acidic determinants; and (5) Abasic residue at the N-terminal of the acceptor site decreases thephosphorylation rate, while an acidic one will increase it.

A consensus pattern for casein kinase II phosphorylations site is asfollows: [ST]-x(2)-[DE], wherein ‘x’ represents any amino acid, and S orT is the phosphorylation site.

Additional information specific to casein kinase II phosphorylationsites may be found in reference to the following publication: Pinna L.A., Biochim. Biophys. Acta 1054:267-284(1990); which is herebyincorporated herein in its entirety.

In preferred embodiments, the following casein kinase II phosphorylationsite polypeptides are encompassed by the present invention:TDGYLSSIESTNLS (SEQ ID NO:29), QISSSSLTDHTFSK (SEQ ID NO:30),LRLSETNCDSKAPN (SEQ ID NO:31), NLCCRTFDEPLCAH (SEQ ID NO:32),MSTVGSRKEMPLLQ (SEQ ID NO:33), and/or SHQATSASESATLD (SEQ ID NO:34).Polynucleotides encoding these polypeptides are also provided. Thepresent invention also encompasses the use of these casein kinase IIphosphorylation site polypeptides as a immunogenic and/or antigenicepitopes as described elsewhere herein.

The HLLRCR-1 polypeptide has been shown to comprise five glycosylationsite according to the Motif algorithm (Genetics Computer Group, Inc.).As discussed more specifically herein, protein glycosylation is thoughtto serve a variety of functions including: augmentation of proteinfolding, inhibition of protein aggregation, regulation of intracellulartrafficking to organelles, increasing resistance to proteolysis,modulation of protein antigenicity, and mediation of intercellularadhesion.

Asparagine phosphorylation sites have the following consensus pattern,N-{P}-[ST]-{P}, wherein N represents the glycosylation site. However, itis well known that that potential N-glycosylation sites are specific tothe consensus sequence Asn-Xaa-Ser/Thr. However, the presence of theconsensus tripeptide is not sufficient to conclude that an asparagineresidue is glycosylated, due to the fact that the folding of the proteinplays an important role in the regulation of N-glycosylation. It hasbeen shown that the presence of proline between Asn and Ser/Thr willinhibit N-glycosylation; this has been confirmed by a recent statisticalanalysis of glycosylation sites, which also shows that about 50% of thesites that have a proline C-terminal to Ser/Thr are not glycosylated.Additional information relating to asparagine glycosylation may be foundin reference to the following publications, which are herebyincorporated by reference herein: Marshall R. D., Annu. Rev. Biochem.41:673-702(1972); Pless D. D., Lennarz W. J., Proc. Natl. Acad. Sci.U.S.A. 74:134-138(1977); Bause E., Biochem. J. 209:331-336(1983); GavelY., von Heijne G., Protein Eng. 3:433-442(1990); and Miletich J. P.,Broze G. J. Jr., J. Biol. Chem. 265:11397-11404(1990).

In preferred embodiments, the following HLLRCR-1 asparagineglycosylation site polypeptides are encompassed by the presentinvention: SIESTNLSLLFNLA (SEQ ID NO:35), GSWFRNTSGLTRLQ (SEQ ID NO:36),GNQITNLTDSSFGG (SEQ ID NO:37), DSKAPNFTLVLKDR (SEQ ID NO:38), and/orEKRLTNESWQPPIE (SEQ ID NO:39). Polynucleotides encoding thesepolypeptides are also provided. The present invention also encompassesthe use of these HLLRCR-1 asparagine glycosylation site polypeptides asimmunogenic and/or antigenic epitopes as described elsewhere herein.

The HLLRCR-1 polypeptide was predicted to comprise two N-myristoylationsites using the Motif algorithm (Genetics Computer Group, Inc.). Anappreciable number of eukaryotic proteins are acylated by the covalentaddition of myristate (a C14-saturated fatty acid) to their N-terminalresidue via an amide linkage. The sequence specificity of the enzymeresponsible for this modification, myristoyl CoA:protein N-myristoyltransferase (NMT), has been derived from the sequence of knownN-myristoylated proteins and from studies using synthetic peptides. Thespecificity seems to be the following: i.) The N-terminal residue mustbe glycine; ii.) In position 2, uncharged residues are allowed; iii.)Charged residues, proline and large hydrophobic residues are notallowed; iv.) In positions 3 and 4, most, if not all, residues areallowed; v.) In position 5, small uncharged residues are allowed (Ala,Ser, Thr, Cys, Asn and Gly). Serine is favored; and vi.) In position 6,proline is not allowed.

A consensus pattern for N-myristoylation is as follows:G-{EDRKHPFYW}-x(2)-[STAGCN]-{P}, wherein ‘x’ represents any amino acid,and G is the N-myristoylation site.

Additional information specific to N-myristoylation sites may be foundin reference to the following publication: Towler D. A., Gordon J. I.,Adams S. P., Glaser L., Annu. Rev. Biochem. 57:69-99(1988); and Grand R.J. A., Biochem. J. 258:625-638(1989); which is hereby incorporatedherein in its entirety.

In preferred embodiments, the following N-myristoylation sitepolypeptides are encompassed by the present invention: RLQLDGNQITNLTDSS(SEQ ID NO:40), SATLDGSFRNLKKKDR (SEQ ID NO:41), KKKDRGVGSTLFCQDG (SEQID NO:42), CSEPPGNMRAFNEAGL (SEQ ID NO:43), and/or AFNEAGLLTTYNPRKV (SEQID NO:44). Polynucleotides encoding these polypeptides are alsoprovided. The present invention also encompasses the use of theseN-myristoylation site polypeptides as immunogenic and/or antigenicepitopes as described elsewhere herein.

The present invention encompasses the identification of compounds anddrugs which stimulate HLLRCR-1 on the one hand (i.e., agonists) andwhich inhibit the function of HLLRCR-1 on the other hand (i.e.,antagonists). In general, such screening procedures involve providingappropriate cells which express the polypeptide of the presentinvention. Such cells may include, for example, cells from mammals,yeast, Drosophila or E. coli. In a preferred embodiment, apolynucleotide encoding the polypeptide of the present invention may beemployed to transfect cells to thereby express the HLLRCR-1 polypeptide.The expressed protein may then be contacted with a test compound toobserve binding, stimulation or inhibition of a functional response.

Many polynucleotide sequences, such as EST sequences, are publiclyavailable and accessible through sequence databases. Some of thesesequences are related to SEQ ID NO: 1 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence would be cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides consisting of a nucleotide sequence described bythe general formula of a-b, where a is any integer between 1 to 1860 ofSEQ ID NO:1, b is an integer between 15 to 1874, where both a and bcorrespond to the positions of nucleotide residues shown in SEQ ID NO:1,and where b is greater than or equal to a+14. TABLE I ATCC Deposit GeneNo: PTA-3434 NT SEQ ID. Total NT Seq 5′ NT of Start 3′ NT AA Seq TotalAA No. CDNA CloneID and Date Vector No. X of Clone Codon of ORF of ORFID No. Y of ORF 1. HLLRCR-1- PTA-3434 PSport1 1 1874 101 1873 2 592(also known Jun. 07, 2001 as GPCR111, GPCR111-80b, and/or HLLRCAR-1) 1.HLLRCR-1b PTA-3434 PSport1 3 1551 1 1551 4 517 Jun. 07, 2001

Table I summarizes the information corresponding to each “Gene No.”described above. The nucleotide sequence identified as “NT SEQ ID NO:X”was assembled from partially homologous (“overlapping”) sequencesobtained from the “cDNA clone ID” identified in Table I and, in somecases, from additional related DNA clones. The overlapping sequenceswere assembled into a single contiguous sequence of high redundancy(usually several overlapping sequences at each nucleotide position),resulting in a final sequence identified as SEQ ID NO:1, and/or SEQ IDNO:34.

The cDNA Clone ID was deposited on the date and given the correspondingdeposit number listed in “ATCC Deposit No:PTA-3434 and Date.” “Vector”refers to the type of vector contained in the cDNA Clone ID.

“Total NT Seq. Of Clone” refers to the total number of nucleotides inthe clone contig identified by “Gene No.” The deposited clone maycontain all or most of the sequence of SEQ ID NO:1 or SEQ ID NO:34. Thenucleotide position of SEQ ID NO:1 or SEQ ID NO:34 of the putative startcodon (methionine) is identified as “5′ NT of Start Codon of ORF.”

The translated amino acid sequence, beginning with the methionine, isidentified as “AA SEQ ID NO:Y” although other reading frames can also beeasily translated using known molecular biology techniques. Thepolypeptides produced by these alternative open reading frames arespecifically contemplated by the present invention.

The total number of amino acids within the open reading frame of SEQ IDNO:2 or SEQ ID NO:35 is identified as “Total AA of ORF”.

SEQ ID NO:1 (where X may be any of the polynucleotide sequencesdisclosed in the sequence listing) and the translated SEQ ID NO:2 (whereY may be any of the polypeptide sequences disclosed in the sequencelisting) are sufficiently accurate and otherwise suitable for a varietyof uses well known in the art and described further herein. Forinstance, SEQ ID NO:1 is useful for designing nucleic acid hybridizationprobes that will detect nucleic acid sequences contained in SEQ ID NO:1or the cDNA contained in the deposited clone. These probes will alsohybridize to nucleic acid molecules in biological samples, therebyenabling a variety of forensic and diagnostic methods of the invention.Similarly, polypeptides identified from SEQ ID NO:2 may be used, forexample, to generate antibodies which bind specifically to proteinscontaining the polypeptides and the proteins encoded by the cDNA clonesidentified in Table I.

SEQ ID NO:3 (where X may be any of the polynucleotide sequencesdisclosed in the sequence listing) and the translated SEQ ID NO:4 (whereY may be any of the polypeptide sequences disclosed in the sequencelisting) are sufficiently accurate and otherwise suitable for a varietyof uses well known in the art and described further herein. Forinstance, SEQ ID NO:3 is useful for designing nucleic acid hybridizationprobes that will detect nucleic acid sequences contained in SEQ ID NO:3or the cDNA contained in the deposited clone. These probes will alsohybridize to nucleic acid molecules in biological samples, therebyenabling a variety of forensic and diagnostic methods of the invention.Similarly, polypeptides identified from SEQ ID NO:4 may be used, forexample, to generate antibodies which bind specifically to proteinscontaining the polypeptides and the proteins encoded by the cDNA clonesidentified in Table I.

Nevertheless, DNA sequences generated by sequencing reactions cancontain sequencing errors. The errors exist as misidentifiednucleotides, or as insertions or deletions of nucleotides in thegenerated DNA sequence. The erroneously inserted or deleted nucleotidesmay cause frame shifts in the reading frames of the predicted amino acidsequence. In these cases, the predicted amino acid sequence divergesfrom the actual amino acid sequence, even though the generated DNAsequence may be greater than 99.9% identical to the actual DNA sequence(for example, one base insertion or deletion in an open reading frame ofover 1000 bases).

Accordingly, for those applications requiring precision in thenucleotide sequence or the amino acid sequence, the present inventionprovides not only the generated nucleotide sequence identified as SEQ IDNO:1 and/or 3 and the predicted translated amino acid sequenceidentified as SEQ ID NO:2 and/or 4, but also a sample of plasmid DNAcontaining a cDNA of the invention deposited with the ATCC, as set forthin Table I. The nucleotide sequence of each deposited clone can readilybe determined by sequencing the deposited clone in accordance with knownmethods. The predicted amino acid sequence can then be verified fromsuch deposits. Moreover, the amino acid sequence of the protein encodedby a particular clone can also be directly determined by peptidesequencing or by expressing the protein in a suitable host cellcontaining the deposited cDNA, collecting the protein, and determiningits sequence.

Accordingly, for those applications requiring precision in thenucleotide sequence or the amino acid sequence, the present inventionprovides not only the generated nucleotide sequence identified as SEQ IDNO:3 and the predicted translated amino acid sequence identified as SEQID NO:4, but also a sample of plasmid DNA containing a cDNA of theinvention deposited with the ATCC, as set forth in Table I. Thenucleotide sequence of each deposited clone can readily be determined bysequencing the deposited clone in accordance with known methods. Thepredicted amino acid sequence can then be verified from such deposits.Moreover, the amino acid sequence of the protein encoded by a particularclone can also be directly determined by peptide sequencing or byexpressing the protein in a suitable host cell containing the depositedcDNA, collecting the protein, and determining its sequence.

The present invention also relates to the genes corresponding to SEQ IDNO:1 and/or 3, SEQ ID NO:2 and/or 4, SEQ ID NO:3, SEQ ID NO:4 and/or thedeposited clone. The corresponding gene can be isolated in accordancewith known methods using the sequence information disclosed herein. Suchmethods include preparing probes or primers from the disclosed sequenceand identifying or amplifying the corresponding gene from appropriatesources of genomic material.

Also provided in the present invention are species homologs, allelicvariants, and/or orthologs. The skilled artisan could, using procedureswell-known in the art, obtain the polynucleotide sequence correspondingto full-length genes (including, but not limited to the full-lengthcoding region), allelic variants, splice variants, orthologs, and/orspecies homologues of genes corresponding to SEQ ID NO:1 and/or 3, SEQID NO:2 and/or 4, SEQ ID NO:3, SEQ ID NO:4 and/or a deposited clone,relying on the sequence from the sequences disclosed herein or theclones deposited with the ATCC. For example, allelic variants and/orspecies homologues may be isolated and identified by making suitableprobes or primers which correspond to the 5′, 3′, or internal regions ofthe sequences provided herein and screening a suitable nucleic acidsource for allelic variants and/or the desired homologue.

The polypeptides of the invention can be prepared in any suitablemanner. Such polypeptides include isolated naturally occurringpolypeptides, recombinantly produced polypeptides, syntheticallyproduced polypeptides, or polypeptides produced by a combination ofthese methods. Means for preparing such polypeptides are well understoodin the art.

The polypeptides may be in the form of the protein, or may be a part ofa larger protein, such as a fusion protein (see below). It is oftenadvantageous to include an additional amino acid sequence which containssecretory or leader sequences, pro-sequences, sequences which aid inpurification, such as multiple histidine residues, or an additionalsequence for stability during recombinant production.

The polypeptides of the present invention are preferably provided in anisolated form, and preferably are substantially purified. Arecombinantly produced version of a polypeptide, can be substantiallypurified using techniques described herein or otherwise known in theart, such as, for example, by the one-step method described in Smith andJohnson, Gene 67:31-40 (1988). Polypeptides of the invention also can bepurified from natural, synthetic or recombinant sources using protocolsdescribed herein or otherwise known in the art, such as, for example,antibodies of the invention raised against the full-length form of theprotein.

The present invention provides a polynucleotide comprising, oralternatively consisting of, the sequence identified as SEQ ID NO:1and/or 3, and/or a cDNA provided in ATCC Deposit No:PTA-3434. Thepresent invention also provides a polypeptide comprising, oralternatively consisting of, the sequence identified as SEQ ID NO:2and/or 4, and/or a polypeptide encoded by the cDNA provided in ATCCDeposit No:PTA-3434. The present invention also provides polynucleotidesencoding a polypeptide comprising, or alternatively consisting of thepolypeptide sequence of SEQ ID NO:2 and/or 4, and/or a polypeptidesequence encoded by the cDNA contained in ATCC Deposit No:PTA-3434.

The present invention provides a polynucleotide comprising, oralternatively consisting of, the sequence identified as SEQ ID NO:3,and/or a cDNA provided in ATCC Deposit No:PTA-3434. The presentinvention also provides a polypeptide comprising, or alternativelyconsisting of, the sequence identified as SEQ ID NO:4, and/or apolypeptide encoded by the cDNA provided in ATCC Deposit No:PTA-3434.The present invention also provides polynucleotides encoding apolypeptide comprising, or alternatively consisting of the polypeptidesequence of SEQ ID NO:4, and/or a polypeptide sequence encoded by thecDNA contained in ATCC Deposit No:PTA-3434.

Preferably, the present invention is directed to a polynucleotidecomprising, or alternatively consisting of, the sequence identified asSEQ ID NO:1 and/or 3, and/or a cDNA provided in ATCC Deposit No:PTA-3434that is less than, or equal to, a polynucleotide sequence that is 5 megabasepairs, 1 mega basepairs, 0.5 mega basepairs, 0.1 mega basepairs,50,000 basepairs, 20,000 basepairs, or 10,000 basepairs in length.

Preferably, the present invention is directed to a polynucleotidecomprising, or alternatively consisting of, the sequence identified asSEQ ID NO:3, and/or a cDNA provided in ATCC Deposit No:PTA-3434 that isless than, or equal to, a polynucleotide sequence that is 5 megabasepairs, 1 mega basepairs, 0.5 mega basepairs, 0.1 mega-basepairs,50,000 basepairs, 20,000 basepairs, or 10,000 basepairs in length.

The present invention encompasses polynucleotides with sequencescomplementary to those of the polynucleotides of the present inventiondisclosed herein. Such sequences may be complementary to the sequencedisclosed as SEQ ID NO:1 and/or 3, the sequence contained in a deposit,and/or the nucleic acid sequence encoding the sequence disclosed as SEQID NO:2 and/or 4.

The present invention encompasses polynucleotides with sequencescomplementary to those of the polynucleotides of the present inventiondisclosed herein. Such sequences may be complementary to the sequencedisclosed as SEQ ID NO:3, the sequence contained in a deposit, and/orthe nucleic acid sequence encoding the sequence disclosed as SEQ IDNO:4.

The present invention also encompasses polynucleotides capable ofhybridizing, preferably under reduced stringency conditions, morepreferably under stringent conditions, and most preferably under highlystringent conditions, to polynucleotides described herein. Examples ofstringency conditions are shown in Table II below: highly stringentconditions are those that are at least as stringent as, for example,conditions A-F; stringent conditions are at least as stringent as, forexample, conditions G-L; and reduced stringency conditions are at leastas stringent as, for example, conditions M-R. TABLE II Strin- Poly-gency nucleo- Hybrid Hybridization Wash Con- tide Length TemperatureTemperature dition Hybrid± (bp) ‡ and Buffer † and Buffer † A DNA:DNA >or equal 65° C.; 1 × SSC -or- 65° C.; 0.3 × SSC to 50 42° C.; 1 × SSC,50% formamide B DNA:DNA <50 Tb*; 1 × SSC Tb*; 1 × SSC C DNA:RNA > orequal 67° C.; 1 × SSC -or- 67° C.; 0.3 × SSC to 50 45° C.; 1 × SSC, 50%formamide D DNA:RNA <50 Td*; 1 × SSC Td*; 1 × SSC E RNA:RNA > or equal70° C.; 1 × SSC -or- 70° C.; 0.3 × SSC to 50 50° C.; 1 × SSC, 50%formamide F RNA:RNA <50 Tf*; 1 × SSC Tf*; 1 × SSC G DNA:DNA > or equal65° C.; 4 × SSC -or- 65° C.; 1 × SSC to 50 45° C.; 4 × SSC, 50%formamide H DNA:DNA <50 Th*; 4 × SSC Th*; 4 × SSC I DNA:RNA > or equal67° C.; 4 × SSC -or- 67° C.; 1 × SSC to 50 45° C.; 4 × SSC, 50%formamide J DNA:RNA <50 Tj*; 4 × SSC Tj*; 4 × SSC K RNA:RNA > or equal70° C.; 4 × SSC -or- 67° C.; 1 × SSC to 50 40° C.; 6 × SSC, 50%formamide L RNA:RNA <50 Tl*; 2 × SSC Tl*; 2 × SSC M DNA:DNA > or equal50° C.; 4 × SSC -or- 50° C.; 2 × SSC to 50 40° C. 6 × SSC, 50% formamideN DNA:DNA <50 Tn*; 6 × SSC Tn*; 6 × SSC O DNA:RNA > or equal 55° C.; 4 ×SSC -or- 55° C.; 2 × SSC to 50 42° C.; 6 × SSC, 50% formamide P DNA:RNA<50 Tp*; 6 × SSC Tp*; 6 × SSC Q RNA:RNA > or equal 60° C.; 4 × SSC -or-60° C.; 2 × SSC to 50 45° C.; 6 × SSC, 50% formamide R RNA:RNA <50 Tr*;4 × SSC Tr*; 4 × SSC‡ - The “hybrid length” is the anticipated length for the hybridizedregion(s) of the hybridizing# polynucleotides. When hybridizing a polynucleotide of unknownsequence, the hybrid is assumed to be that of the # hybridizingpolynucleotide of the present invention. When polynucleotides of known #sequence are hybridized, the hybrid length can be determined by aligningthe sequences of the polynucleotides # and identifying the region orregions of optimal sequence complementarity. Methods of aligning two ormore # polynucleotide sequences and/or determining the percent identitybetween two polynucleotide # sequences are well known in the art (e.g.,MegAlign program of the DNA*Star suite of programs, etc).† - SSPE (1 × SSPE is 0.15 M NaCl, 10 mM NaH2PO4, and 1.25 mM EDTA, pH7.4) can be substituted for# SSC (1 × SSC is 0.15 M NaCl and 15 mM sodium citrate) in thehybridization and wash buffers; washes are # performed for 15 minutesafter hybridization is complete. The hydridizations and washes # mayadditionally include 5× Denhardt's reagent, .5-1.0% SDS, 100 ug/mldenatured, fragmented salmon # sperm DNA, 0.5% sodium pyrophosphate, andup to 50% formamide.

*Tb-Tr: The hybridization temperature for hybrids anticipated to be lessthan 50 base pairs in length should be 5-10° C. less than the meltingtemperature Tm of the hybrids there Tm is determined according to thefollowing equations. For hybrids less than 18 base pairs in length, Tm(°C.)=2(# of A+T bases)+4(# of G+C bases). For hybrids between 18 and 49base pairs in length, Tm(° C.)=81.5+16.6(log₁₀[Na+])+0.41(%G+C)−(600/N),where N is the number of bases in the hybrid, and [Na+] is theconcentration of sodium ions in the hybridization buffer ([NA+] for1×SSC=0.165 M).

±—The present invention encompasses the substitution of any one, or moreDNA or RNA hybrid partners with either a PNA, or a modifiedpolynucleotide. Such modified polynucleotides are known in the art andare more particularly described elsewhere herein.

Additional examples of stringency conditions for polynucleotidehybridization are provided, for example, in Sambrook, J., E. F. Fritsch,and T. Maniatis, 1989, Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y., chapters 9 and11, and Current Protocols in Molecular Biology, 1995, F. M., Ausubel etal., eds, John Wiley and Sons, Inc., sections 2.10 and 6.3-6.4, whichare hereby incorporated by reference herein.

Preferably, such hybridizing polynucleotides have at least 70% sequenceidentity (more preferably, at least 80% identity; and most preferably atleast 90% or 95% identity) with the polynucleotide of the presentinvention to which they hybridize, where sequence identity is determinedby comparing the sequences of the hybridizing polynucleotides whenaligned so as to maximize overlap and identity while minimizing sequencegaps. The determination of identity is well known in the art, anddiscussed more specifically elsewhere herein.

The invention encompasses the application of PCR methodology to thepolynucleotide sequences of the present invention, the clone depositedwith the ATCC, and/or the cDNA encoding the polypeptides of the presentinvention. PCR techniques for the amplification of nucleic acids aredescribed in U.S. Pat. No. 4,683,195 and Saiki et al., Science,239:487-491 (1988). PCR, for example, may include the following steps,of denaturation of template nucleic acid (if double-stranded), annealingof primer to target, and polymerization. The nucleic acid probed or usedas a template in the amplification reaction may be genomic DNA, cDNA,RNA, or a PNA. PCR may be used to amplify specific sequences fromgenomic DNA, specific RNA sequence, and/or cDNA transcribed from mRNA.References for the general use of PCR techniques, including specificmethod parameters, include Mullis et al., Cold Spring Harbor Symp.Quant. Biol., 51:263, (1987), Ehrlich (ed), PCR Technology, StocktonPress, NY, 1989; Ehrlich et al., Science, 252:1643-1650, (1991); and“PCR Protocols, A Guide to Methods and Applications”, Eds., Innis etal., Academic Press, New York, (1990).

Signal Sequences

The present invention also encompasses mature forms of the polypeptidecomprising, or alternatively consisting of, the polypeptide sequence ofSEQ ID NO:2, the polypeptide encoded by the polynucleotide described asSEQ ID NO:1, and/or the polypeptide sequence encoded by a cDNA in thedeposited clone. The present invention also encompasses polynucleotidesencoding mature forms of the present invention, such as, for example thepolynucleotide sequence of SEQ ID NO:1, and/or the polynucleotidesequence provided in a cDNA of the deposited clone.

According to the signal hypothesis, proteins secreted by eukaryoticcells have a signal or secretary leader sequence which is cleaved fromthe mature protein once export of the growing protein chain across therough endoplasmic reticulum has been initiated. Most eukaryotic cellscleave secreted proteins with the same specificity. However, in somecases, cleavage of a secreted protein is not entirely uniform, whichresults in two or more mature species of the protein. Further, it haslong been known that cleavage specificity of a secreted protein isultimately determined by the primary structure of the complete protein,that is, it is inherent in the amino acid sequence of the polypeptide.

Methods for predicting whether a protein has a signal sequence, as wellas the cleavage point for that sequence, are available. For instance,the method of McGeoch, Virus Res. 3:271-286 (1985), uses the informationfrom a short N-terminal charged region and a subsequent uncharged regionof the complete (uncleaved) protein. The method of von Heinje, NucleicAcids Res. 14:4683-4690 (1986) uses the information from the residuessurrounding the cleavage site, typically residues −13 to +2, where +1indicates the amino terminus of the secreted protein. The accuracy ofpredicting the cleavage points of known mammalian secretory proteins foreach of these methods is in the range of 75-80%. (von Heinje, supra.)However, the two methods do not always produce the same predictedcleavage point(s) for a given protein.

The established method for identifying the location of signal sequences,in addition, to their cleavage sites has been the SignalP program (v1.1)developed by Henrik Nielsen et al., Protein Engineering 10:1-6 (1997).The program relies upon the algorithm developed by von Heinje, thoughprovides additional parameters to increase the prediction accuracy.

More recently, a hidden Markov model has been developed (H. Neilson, etal., Ismb 1998;6:122-30), which has been incorporated into the morerecent SignalP (v2.0). This new method increases the ability to identifythe cleavage site by discriminating between signal peptides anduncleaved signal anchors. The present invention encompasses theapplication of the method disclosed therein to the prediction of thesignal peptide location, including the cleavage site, to any of thepolypeptide sequences of the present invention.

As one of ordinary skill would appreciate, however, cleavage sitessometimes vary from organism to organism and cannot be predicted withabsolute certainty. Accordingly, the polypeptide of the presentinvention may contain a signal sequence. Polypeptides of the inventionwhich comprise a signal sequence have an N-terminus beginning within 5residues (i.e., +or −5 residues, or Preferably at the −5, −4, −3, −2,−1, +1, +2, +3, +4, or +5 residue) of the predicted cleavage point.Similarly, it is also recognized that in some cases, cleavage of thesignal sequence from a secreted protein is not entirely uniform,resulting in more than one secreted species. These polypeptides, and thepolynucleotides encoding such polypeptides, are contemplated by thepresent invention.

Moreover, the signal sequence identified by the above analysis may notnecessarily predict the naturally occurring signal sequence. Forexample, the naturally occurring signal sequence may be further upstreamfrom the predicted signal sequence. However, it is likely that thepredicted signal sequence will be capable of directing the secretedprotein to the ER. Nonetheless, the present invention provides themature protein produced by expression of the polynucleotide sequence ofSEQ ID NO:1 and/or the polynucleotide sequence contained in the cDNA ofa deposited clone, in a mammalian cell (e.g., COS cells, as desribedbelow). These polypeptides, and the polynucleotides encoding suchpolypeptides, are contemplated by the present invention.

Polynucleotide and Polypeptide Variants

The present invention also encompasses variants (e.g., allelic variants,orthologs, etc.) of the polynucleotide sequence disclosed herein in SEQID NO:1 and/or 3, the complementary strand thereto, and/or the cDNAsequence contained in the deposited clone.

The present invention also encompasses variants (e.g., allelic variants,orthologs, etc.) of the polynucleotide sequence disclosed herein in SEQID NO:3, the complementary strand thereto, and/or the cDNA sequencecontained in the deposited clone.

The present invention also encompasses variants of the polypeptidesequence, and/or fragments therein, disclosed in SEQ ID NO:2 and/or 4, apolypeptide encoded by the polynucleotide sequence in SEQ ID NO:1 and/or3, and/or a polypeptide encoded by a cDNA in the deposited clone.

The present invention also encompasses variants of the polypeptidesequence, and/or fragments therein, disclosed in SEQ ID NO:4, apolypeptide encoded by the polynucleotide sequence in SEQ ID NO:3,and/or a polypeptide encoded by a cDNA in the deposited clone.

“Variant” refers to a polynucleotide or polypeptide differing from thepolynucleotide or polypeptide of the present invention, but retainingessential properties thereof. Generally, variants are overall closelysimilar, and, in many regions, identical to the polynucleotide orpolypeptide of the present invention.

Thus, one aspect of the invention provides an isolated nucleic acidmolecule comprising, or alternatively consisting of, a polynucleotidehaving a nucleotide sequence selected from the group consisting of: (a)a nucleotide sequence encoding a HLLRCR-1 related polypeptide having anamino acid sequence as shown in the sequence listing and described inSEQ ID NO:1 and/or 3 or the cDNA contained in ATCC deposit No:PTA-3434;(b) a nucleotide sequence encoding a mature HLLRCR-1 related polypeptidehaving the amino acid sequence as shown in the sequence listing anddescribed in SEQ ID NO:1 and/or 3 or the cDNA contained in ATCC depositNo:PTA-3434; (c) a nucleotide sequence encoding a biologically activefragment of a HLLRCR-1 related polypeptide having an amino acid sequenceshown in the sequence listing and described in SEQ ID NO:1 and/or 3 orthe cDNA contained in ATCC deposit No:PTA-3434; (d) a nucleotidesequence encoding an antigenic fragment of a HLLRCR-1 relatedpolypeptide having an amino acid sequence sown in the sequence listingand described in SEQ ID NO:1 and/or 3 or the cDNA contained in ATCCdeposit No:PTA-3434; (e) a nucleotide sequence encoding a HLLRCR-1related polypeptide comprising the complete amino acid sequence encodedby a human cDNA plasmid contained in SEQ ID NO:1 and/or 3 or the cDNAcontained in ATCC deposit No:PTA-3434; (f) a nucleotide sequenceencoding a mature HLLRCR-1 related polypeptide having an amino acidsequence encoded by a human cDNA plasmid contained in SEQ ID NO:1 and/or3 or the cDNA contained in ATCC deposit No:PTA-3434; (g) a nucleotidesequence encoding a biologically active fragment of a HLLRCR-1 relatedpolypeptide having an amino acid sequence encoded by a human cDNAplasmid contained in SEQ ID NO:1 and/or 3 or the cDNA contained in ATCCdeposit No:PTA-3434; (h) a nucleotide sequence encoding an antigenicfragment of a HLLRCR-1 related polypeptide having an amino acid sequenceencoded by a human cDNA plasmid contained in SEQ ID NO:1 and/or 3 or thecDNA contained in ATCC deposit No:PTA-3434; (I) a nucleotide sequencecomplimentary to any of the nucleotide sequences in (a), (b), (c), (d),(e), (f), (g), or (h), above.

Thus, one aspect of the invention provides an isolated nucleic acidmolecule comprising, or alternatively consisting of, a polynucleotidehaving a nucleotide sequence selected from the group consisting of: (a)a nucleotide sequence encoding a HLLRCR-1 related polypeptide having anamino acid sequence as shown in the sequence listing and described inSEQ ID NO:3 or the cDNA contained in ATCC deposit No:PTA-3434; (b) anucleotide sequence encoding a mature HLLRCR-1 related polypeptidehaving the amino acid sequence as shown in the sequence listing anddescribed in SEQ ID NO:3 or the cDNA contained in ATCC depositNo:PTA-3434; (c) a nucleotide sequence encoding a biologically activefragment of a HLLRCR-1 related polypeptide having an amino acid sequenceshown in the sequence listing and described in SEQ ID NO:3 or the cDNAcontained in ATCC deposit No:PTA-3434; (d) a nucleotide sequenceencoding an antigenic fragment of a HLLRCR-1 related polypeptide havingan amino acid sequence sown in the sequence listing and described in SEQID NO:3 or the cDNA contained in ATCC deposit No:PTA-3434; (e) anucleotide sequence encoding a HLLRCR-1 related polypeptide comprisingthe complete amino acid sequence encoded by a human cDNA plasmidcontained in SEQ ID NO:3 or the cDNA contained in ATCC depositNo:PTA-3434; (f) a nucleotide sequence encoding a mature HLLRCR-1related polypeptide having an amino acid sequence encoded by a humancDNA plasmid contained in SEQ ID NO:3 or the cDNA contained in ATCCdeposit No:PTA-3434; (g) a nucleotide sequence encoding a biologicallyactive fragment of a HLLRCR-1 related polypeptide having an amino acidsequence encoded by a human cDNA plasmid contained in SEQ ID NO:3 or thecDNA contained in ATCC deposit No:PTA-3434; (h) a nucleotide sequenceencoding an antigenic fragment of a HLLRCR-1 related polypeptide havingan amino acid sequence encoded by a human cDNA plasmid contained in SEQID NO:3 or the cDNA contained in ATCC deposit No:PTA-3434; (I) anucleotide sequence complimentary to any of the nucleotide sequences in(a), (b), (c), (d), (e), (f), (g), or (h), above.

The present invention is also directed to polynucleotide sequences whichcomprise, or alternatively consist of, a polynucleotide sequence whichis at least about 80%, 83%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, 99.1% 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or99.9% identical to, for example, any of the nucleotide sequences in (a),(b), (c), (d), (e), (f), (g), or (h), above. Polynucleotides encoded bythese nucleic acid molecules are also encompassed by the invention. Inanother embodiment, the invention encompasses nucleic acid moleculeswhich comprise, or alternatively, consist of a polynucleotide whichhybridizes under stringent conditions, or alternatively, under lowerstringency conditions, to a polynucleotide in (a), (b), (c), (d), (e),(f), (g), or (h), above. Polynucleotides which hybridize to thecomplement of these nucleic acid molecules under stringent hybridizationconditions or alternatively, under lower stringency conditions, are alsoencompassed by the invention, as are polypeptides encoded by thesepolypeptides.

Another aspect of the invention provides an isolated nucleic acidmolecule comprising, or alternatively, consisting of, a polynucleotidehaving a nucleotide sequence selected from the group consisting of: (a)a nucleotide sequence encoding a HLLRCR-1 related polypeptide having anamino acid sequence as shown in the sequence listing and descried inTable I; (b) a nucleotide sequence encoding a mature HLLRCR-1 relatedpolypeptide having the amino acid sequence as shown in the sequencelisting and descried in Table I; (c) a nucleotide sequence encoding abiologically active fragment of a HLLRCR-1 related polypeptide having anamino acid sequence as shown in the sequence listing and descried inTable I; (d) a nucleotide sequence encoding an antigenic fragment of aHLLRCR-1 related polypeptide having an amino acid sequence as shown inthe sequence listing and descried in Table I; (e) a nucleotide sequenceencoding a HLLRCR-1 related polypeptide comprising the complete aminoacid sequence encoded by a human cDNA in a cDNA plasmid contained in theATCC Deposit and described in Table I; (f) a nucleotide sequenceencoding a mature HLLRCR-1 related polypeptide having an amino acidsequence encoded by a human cDNA in a cDNA plasmid contained in the ATCCDeposit and described in Table I: (g) a nucleotide sequence encoding abiologically active fragment of a HLLRCR-1 related polypeptide having anamino acid sequence encoded by a human cDNA in a cDNA plasmid containedin the ATCC Deposit and described in Table I; (h) a nucleotide sequenceencoding an antigenic fragment of a HLLRCR-1 related polypeptide havingan amino acid sequence encoded by a human cDNA in a cDNA plasmidcontained in the ATCC deposit and described in Table I; (i) a nucleotidesequence complimentary to any of the nucleotide sequences in (a), (b),(c), (d), (e), (f), (g), or (h) above.

The present invention is also directed to nucleic acid molecules whichcomprise, or alternatively, consist of, a nucleotide sequence which isat least about 80%, 83%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, 99.1% 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or99.9% identical to, for example, any of the nucleotide sequences in (a),(b), (c), (d), (e), (f), (g), or (h), above.

The present invention encompasses polypeptide sequences which comprise,or alternatively consist of, an amino acid sequence which is at leastabout 80%, 81%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9%identical to, the following non-limited examples, the polypeptidesequence identified as SEQ ID NO:2 and/or 4, the polypeptide sequenceencoded by a cDNA provided in the deposited clone, and/or polypeptidefragments of any of the polypeptides provided herein. Polynucleotidesencoded by these nucleic acid molecules are also encompassed by theinvention. In another embodiment, the invention encompasses nucleic acidmolecules which comprise, or alternatively, consist of a polynucleotidewhich hybridizes under stringent conditions, or alternatively, underlower stringency conditions, to a polynucleotide in (a), (b), (c), (d),(e), (f), (g), or (h), above. Polynucleotides which hybridize to thecomplement of these nucleic acid molecules under stringent hybridizationconditions or alternatively, under lower stringency conditions, are alsoencompassed by the invention, as are polypeptides encoded by thesepolypeptides.

The present invention is also directed to polypeptides which comprise,or alternatively consist of, an amino acid sequence which is at leastabout 80%, 81%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9%identical to, for example, the polypeptide sequence shown in SEQ ID NO:2and/or 4, a polypeptide sequence encoded by the nucleotide sequence inSEQ ID NO:1 and/or 3, a polypeptide sequence encoded by the cDNA in ATCCDeposit No:PTA-3434, and/or polypeptide fragments of any of thesepolypeptides (e.g., those fragments described herein). Polynucleotideswhich hybridize to the complement of the nucleic acid molecules encodingthese polypeptides under stringent hybridization conditions oralternatively, under lower stringency conditions, are also encompassesby the present invention, as are the polypeptides encoded by thesepolynucleotides.

The present invention is also directed to polypeptides which comprise,or alternatively consist of, an amino acid sequence which is at leastabout 80%, 81%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9%identical to, for example, the polypeptide sequence shown in SEQ ID NO:2and/or 4, a polypeptide sequence encoded by the nucleotide sequence inSEQ ID NO: 1 and/or 3, a polypeptide sequence encoded by the cDNA inATCC Deposit No:PTA-3434, and/or polypeptide fragments of any of thesepolypeptides (e.g., those fragments described herein). Polynucleotideswhich hybridize to the complement of the nucleic acid molecules encodingthese polypeptides under stringent hybridization conditions oralternatively, under lower stringency conditions, are also encompassesby the present invention, as are the polypeptides encoded by thesepolynucleotides.

By a nucleic acid having a nucleotide sequence at least, for example,95% “identical” to a reference nucleotide sequence of the presentinvention, it is intended that the nucleotide sequence of the nucleicacid is identical to the reference sequence except that the nucleotidesequence may include up to five point mutations per each 100 nucleotidesof the reference nucleotide sequence encoding the polypeptide. In otherwords, to obtain a nucleic acid having a nucleotide sequence at least95% identical to a reference nucleotide sequence, up to 5% of thenucleotides in the reference sequence may be deleted or substituted withanother nucleotide, or a number of nucleotides up to 5% of the totalnucleotides in the reference sequence may be inserted into the referencesequence. The query sequence may be an entire sequence referenced inTable I, the ORF (open reading frame), or any fragment specified asdescribed-herein.

As a practical matter, whether any particular nucleic acid molecule orpolypeptide is at least about 80%, 81%, 83%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%,99.7%, 99.8%, or 99.9% identical to a nucleotide sequence of the presentinvention can be determined conventionally using known computerprograms. A preferred method for determining the best overall matchbetween a query sequence (a sequence of the present invention) and asubject sequence, also referred to as a global sequence alignment, canbe determined using the CLUSTALW computer program (Thompson, J. D., etal., Nucleic Acids Research, 2(22):4673-4680, (1994)), which is based onthe algorithm of Higgins, D. G., et al., Computer Applications in theBiosciences (CABIOS), 8(2):189-191, (1992). In a sequence alignment thequery and subject sequences are both DNA sequences. An RNA sequence canbe compared by converting U's to T's. However, the CLUSTALW algorithmautomatically converts U's to T's when comparing RNA sequences to DNAsequences. The result of said global sequence alignment is in percentidentity. Preferred parameters used in a CLUSTALW alignment of DNAsequences to calculate percent identity via pairwise alignments are:Matrix=IUB, k-tuple=1, Number of Top Diagonals=5, Gap Penalty=3, GapOpen Penalty 10, Gap Extension Penalty=0.1, Scoring Method=Percent,Window Size=5 or the length of the subject nucleotide sequence,whichever is shorter. For multiple alignments, the following CLUSTALWparameters are preferred: Gap Opening Penalty=10; Gap ExtensionParameter=0.05; Gap Separation Penalty Range=8; End Gap SeparationPenalty=Off; % Identity for Alignment Delay=40%; Residue SpecificGaps:Off; Hydrophilic Residue Gap=Off; and Transition Weighting=0. Thepairwise and multple alignment parameters provided for CLUSTALW aboverepresent the default parameters as provided with the AlignX softwareprogram (Vector NTI suite of programs, version 6.0).

The present invention encompasses the application of a manual correctionto the percent identity results, in the instance where the subjectsequence is shorter than the query sequence because of 5′ or 3′deletions, not because of internal deletions. If only the local pairwisepercent identity is required, no manual correction is needed. However, amanual correction may be applied to determine the global percentidentity from a global polynucleotide alignment. Percent identitycalculations based upon global polynucleotide alignments are oftenpreferred since they reflect the percent identity between thepolynucleotide molecules as a whole (i.e., including any polynucleotideoverhangs, not just overlapping regions), as opposed to, only localmatching polynucleotides. Manual corrections for global percent identitydeterminations are required since the CLUSTALW program does not accountfor 5′ and 3′ truncations of the subject sequence when calculatingpercent identity. For subject sequences truncated at the 5′ or 3′ ends,relative to the query sequence, the percent identity is corrected bycalculating the number of bases of the query sequence that are 5′ and 3′of the subject sequence, which are not matched/aligned, as a percent ofthe total bases of the query sequence. Whether a nucleotide ismatched/aligned is determined by results of the CLUSTALW sequencealignment. This percentage is then subtracted from the percent identity,calculated by the above CLUSTALW program using the specified parameters,to arrive at a final percent identity score. This corrected score may beused for the purposes of the present invention. Only bases outside the5′ and 3′ bases of the subject sequence, as displayed by the CLUSTALWalignment, which are not matched/aligned with the query sequence, arecalculated for the purposes of manually adjusting the percent identityscore.

For example, a 90 base subject sequence is aligned to a 100 base querysequence to determine percent identity. The deletions occur at the 5′end of the subject sequence and therefore, the CLUSTALW alignment doesnot show a matched/alignment of the first 10 bases at 5′ end. The 10unpaired bases represent 10% of the sequence (number of bases at the 5′and 3′ ends not matched/total number of bases in the query sequence) so10% is subtracted from the percent identity score calculated by theCLUSTALW program. If the remaining 90 bases were perfectly matched thefinal percent identity would be 90%. In another example, a 90 basesubject sequence is compared with a 100 base query sequence. This timethe deletions are internal deletions so that there are no bases on the5′ or 3′ of the subject sequence which are not matched/aligned with thequery. In this case the percent identity calculated by CLUSTALW is notmanually corrected. Once again, only bases 5′ and 3′ of the subjectsequence which are not matched/aligned with the query sequence aremanually corrected for. No other manual corrections are required for thepurposes of the present invention.

By a polypeptide having an amino acid sequence at least, for example,95% “identical” to a query amino acid sequence of the present invention,it is intended that the amino acid sequence of the subject polypeptideis identical to the query sequence except that the subject polypeptidesequence may include up to five amino acid alterations per each 100amino acids of the query amino acid sequence. In other words, to obtaina polypeptide having an amino acid sequence at least 95% identical to aquery amino acid sequence, up to 5% of the amino acid residues in thesubject sequence may be inserted, deleted, or substituted with anotheramino acid. These alterations of the reference sequence may occur at theamino- or carboxy-terminal positions of the reference amino acidsequence or anywhere between those terminal positions, interspersedeither individually among residues in the reference sequence or in oneor more contiguous groups within the reference sequence.

As a practical matter, whether any particular polypeptide is at leastabout 80%, 81%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9%identical to, for instance, an amino acid sequence referenced in Table 1(SEQ ID NO:2 and/or 4) or to the amino acid sequence encoded by cDNAcontained in a deposited clone, can be determined conventionally usingknown computer programs. A preferred method for determining the bestoverall match between a query sequence (a sequence of the presentinvention) and a subject sequence, also referred to as a global sequencealignment, can be determined using the CLUSTALW computer program(Thompson, J. D., et al., Nucleic Acids Research, 2(22):4673-4680,(1994)), which is based on the algorithm of Higgins, D. G., et al.,Computer Applications in the Biosciences (CABIOS), 8(2):189-191, (1992).In a sequence alignment the query and subject sequences are both aminoacid sequences. The result of said global sequence alignment is inpercent identity. Preferred parameters used in a CLUSTALW alignment ofpolypeptide sequences to calculate percent identity via pairwisealignments are: Matrix=BLOSUM, k-tuple=1, Number of Top Diagonals=5, GapPenalty=3, Gap Open Penalty 10, Gap Extension Penalty=0.1, ScoringMethod=Percent, Window Size=5 or the length of the subject nucleotidesequence, whichever is shorter. For multiple alignments, the followingCLUSTALW parameters are preferred: Gap Opening Penalty=10; Gap ExtensionParameter=0.05; Gap Separation Penalty Range=8; End Gap SeparationPenalty=Off; % Identity for Alignment Delay=40%; Residue SpecificGaps:Off; Hydrophilic Residue Gap=Off; and Transition Weighting=0. Thepairwise and multple alignment parameters provided for CLUSTALW aboverepresent the default parameters as provided with the AlignX softwareprogram (Vector NTI suite of programs, version 6.0).

The present invention encompasses the application of a manual correctionto the percent identity results, in the instance where the subjectsequence is shorter than the query sequence because of N— or C-terminaldeletions, not because of internal deletions. If only the local pairwisepercent identity is required, no manual correction is needed. However, amanual correction may be applied to determine the global percentidentity from a global polypeptide alignment. Percent identitycalculations based upon global polypeptide alignments are oftenpreferred since they reflect the percent identity between thepolypeptide molecules as a whole (i.e., including any polypeptideoverhangs, not just overlapping regions), as opposed to, only localmatching polypeptides. Manual corrections for global percent identitydeterminations are required since the CLUSTALW program does not accountfor N— and C-terminal truncations of the subject sequence whencalculating percent identity. For subject sequences truncated at the N—and C-termini, relative to the query sequence, the percent identity iscorrected by calculating the number of residues of the query sequencethat are N— and C-terminal of the subject sequence, which are notmatched/aligned with a corresponding subject residue, as a percent ofthe total bases of the query sequence. Whether a residue ismatched/aligned is determined by results of the CLUSTALW sequencealignment. This percentage is then subtracted from the percent identity,calculated by the above CLUSTALW program using the specified parameters,to arrive at a final percent identity score. This final percent identityscore is what may be used for the purposes of the present invention.Only residues to the N— and C-termini of the subject sequence, which arenot matched/aligned with the query sequence, are considered for thepurposes of manually adjusting the percent identity score. That is, onlyquery residue positions outside the farthest N— and C-terminal residuesof the subject sequence.

For example, a 90 amino acid residue subject sequence is aligned with a100 residue query sequence to determine percent identity. The deletionoccurs at the N-terminus of the subject sequence and therefore, theCLUSTALW alignment does not show a matching/alignment of the first 10residues at the N-terminus. The 10 unpaired residues represent 10% ofthe sequence (number of residues at the N— and C-termini notmatched/total number of residues in the query sequence) so 10% issubtracted from the percent identity score calculated by the CLUSTALWprogram. If the remaining 90 residues were perfectly matched the finalpercent identity would be 90%. In another example, a 90 residue subjectsequence is compared with a 100 residue query sequence. This time thedeletions are internal deletions so there are no residues at the N— orC-termini of the subject sequence, which are not matched/aligned withthe query. In this case the percent identity calculated by CLUSTALW isnot manually corrected. Once again, only residue positions outside theN— and C-terminal ends of the subject sequence, as displayed in theCLUSTALW alignment, which are not matched/aligned with the querysequence are manually corrected for. No other manual corrections arerequired for the purposes of the present invention.

In addition to the above method of aligning two or more polynucleotideor polypeptide sequences to arrive at a percent identity value for thealigned sequences, it may be desirable in some circumstances to use amodified version of the CLUSTALW algorithm which takes, into accountknown structural features of the sequences to be aligned, such as forexample, the SWISS-PROT designations for each sequence. The result ofsuch a modifed CLUSTALW algorithm may provide a more accurate value ofthe percent identity for two polynucleotide or polypeptide sequences.Support for such a modified version of CLUSTALW is provided within theCLUSTALW algorithm and would be readily appreciated to one of skill inthe art of bioinformatics.

The variants may contain alterations in the coding regions, non-codingregions, or both. Especially preferred are polynucleotide variantscontaining alterations which produce silent substitutions, additions, ordeletions, but do not alter the properties or activities of the encodedpolypeptide. Nucleotide variants produced by silent substitutions due tothe degeneracy of the genetic code are preferred. Moreover, variants inwhich 5-10, 1-5, or 1-2 amino acids are substituted, deleted, or addedin any combination are also preferred. Polynucleotide variants can beproduced for a variety of reasons, e.g., to optimize codon expressionfor a particular host (change codons in the mRNA to those preferred by abacterial host such as E. coli).

Naturally occurring variants are called “allelic variants,” and refer toone of several alternate forms of a gene occupying a given locus on achromosome of an organism. (Genes II, Lewin, B., ed., John Wiley & Sons,New York (1985).) These allelic variants can vary at either thepolynucleotide and/or polypeptide level and are included in the presentinvention. Alternatively, non-naturally occurring variants may beproduced by mutagenesis techniques or by direct synthesis.

Using known methods of protein engineering and recombinant DNAtechnology, variants may be generated to improve or alter thecharacteristics of the polypeptides of the present invention. Forinstance, one or more amino acids can be deleted from the N-terminus orC-terminus of the protein without substantial loss of biologicalfunction. The authors of Ron et al., J. Biol. Chem. 268: 2984-2988(1993), reported variant KGF proteins having heparin binding activityeven after deleting 3, 8, or 27 amino-terminal amino acid residues.Similarly, Interferon gamma exhibited up to ten times higher activityafter deleting 8-10 amino acid residues from the carboxy terminus ofthis protein (Dobeli et al., J. Biotechnology 7:199-216 (1988)).

Moreover, ample evidence demonstrates that variants often retain abiological activity similar to that of the naturally occurring protein.For example, Gayle and coworkers (J. Biol. Chem. 268:22105-22111 (1993))conducted extensive mutational analysis of human cytokine IL-1a. Theyused random mutagenesis to generate over 3,500 individual IL-1a mutantsthat averaged 2.5 amino acid changes per variant over the entire lengthof the molecule. Multiple mutations were examined at every possibleamino acid position. The investigators found that “[m]ost of themolecule could be altered with little effect on either [binding orbiological activity].” In fact, only 23 unique amino acid sequences, outof more than 3,500 nucleotide sequences examined, produced a proteinthat significantly differed in activity from wild-type.

Furthermore, even if deleting one or more amino acids from theN-terminus or C-terminus of a polypeptide results in modification orloss of one or more biological functions, other biological activitiesmay still be retained. For example, the ability of a deletion variant toinduce and/or to bind antibodies which recognize the protein will likelybe retained when less than the majority of the residues of the proteinare removed from the N-terminus or C-terminus. Whether a particularpolypeptide lacking N— or C-terminal residues of a protein retains suchimmunogenic activities can readily be determined by routine methodsdescribed herein and otherwise known in the art.

Alternatively, such N-terminus or C-terminus deletions of a polypeptideof the present invention may, in fact, result in a significant increasein one or more of the biological activities of the polypeptide(s). Forexample, biological activity of many polypeptides are governed by thepresence of regulatory domains at either one or both termini. Suchregulatory domains effectively inhibit the biological activity of suchpolypeptides in lieu of an activation event (e.g., binding to a cognateligand or receptor, phosphorylation, proteolytic processing, etc.).Thus, by eliminating the regulatory domain of a polypeptide, thepolypeptide may effectively be rendered biologically active in theabsence of an activation event.

Thus, the invention further includes polypeptide variants that showsubstantial biological activity. Such variants include deletions,insertions, inversions, repeats, and substitutions selected according togeneral rules known in the art so as have little effect on activity. Forexample, guidance concerning how to make phenotypically silent aminoacid substitutions is provided in Bowie et al., Science 247:1306-1310(1990), wherein the authors indicate that there are two main strategiesfor studying the tolerance of an amino acid sequence to change.

The first strategy exploits the tolerance of amino acid substitutions bynatural selection during the process of evolution. By comparing aminoacid sequences in different species, conserved amino acids can beidentified. These conserved amino acids are likely important for proteinfunction. In contrast, the amino acid positions where substitutions havebeen tolerated by natural selection indicates that these positions arenot critical for protein function. Thus, positions tolerating amino acidsubstitution could be modified while still maintaining biologicalactivity of the protein.

The second strategy uses genetic engineering to introduce amino acidchanges at specific positions of a cloned gene to identify regionscritical for protein function. For example, site directed mutagenesis oralanine-scanning mutagenesis (introduction of single alanine mutationsat every residue in the molecule) can be used. (Cunningham and Wells,Science 244:1081-1085 (1989).) The resulting mutant molecules can thenbe tested for biological activity.

As the authors state, these two strategies have revealed that proteinsare surprisingly tolerant of amino acid substitutions. The authorsfurther indicate which amino acid changes are likely to be permissive atcertain amino acid positions in the protein. For example, most buried(within the tertiary structure of the protein) amino acid residuesrequire nonpolar side chains, whereas few features of surface sidechains are generally conserved.

The invention encompasses polypeptides having a lower degree of identitybut having sufficient similarity so as to perform one or more of thesame functions performed by the polypeptide of the present invention.Similarity is determined by conserved amino acid substitution. Suchsubstitutions are those that substitute a given amino acid in apolypeptide by another amino acid of like characteristics (e.g.,chemical properties). According to Cunningham et al above, suchconservative substitutions are likely to be phenotypically silent.Additional guidance concerning which amino acid changes are likely to bephenotypically silent are found in Bowie et al., Science 247:1306-1310(1990).

Tolerated conservative amino acid substitutions of the present inventioninvolve replacement of the aliphatic or hydrophobic amino acids Ala,Val, Leu and Ile; replacement of the hydroxyl residues Ser and Thr;replacement of the acidic residues Asp and Glu; replacement of the amideresidues Asn and Gln, replacement of the basic residues Lys, Arg, andHis; replacement of the aromatic residues Phe, Tyr, and Trp, andreplacement of the small-sized amino acids Ala, Ser, Thr, Met, and Gly.

In addition, the present invention also encompasses the conservativesubstitutions provided in Table III below. TABLE III For Amino Acid CodeReplace with any of: Alanine A D-Ala, Gly, beta-Ala, L-Cys, D-CysArginine R D-Arg, Lys, D-Lys, homo-Arg, D- homo-Arg, Met, Ile, D-Met, D-Ile, Orn, D-Orn Asparagine N D-Asn, Asp, D-Asp, Glu, D-Glu, Gln, D-GlnAspartic D D-Asp, D-Asn, Asn, Glu, D-Glu, Acid Gln, D-Gln Cysteine CD-Cys, S-Me-Cys, Met, D-Met, Thr, D-Thr Glutamine Q D-Gln, Asn, D-Asn,Glu, D-Glu, Asp, D-Asp Glutamic E D-Glu, D-Asp, Asp, Asn, D-Asn, AcidGln, D-Gln Glycine G Ala, D-Ala, Pro, D-Pro, β-Ala, Acp Isoleucine ID-Ile, Val, D-Val, Leu, D-Leu, Met, D-Met Leucine L D-Leu, Val, D-Val,Met, D-Met Lysine K D-Lys, Arg, D-Arg, homo-Arg, D- homo-Arg, Met,D-Met, Ile, D- Ile, Orn, D-Orn Methionine M D-Met, S-Me-Cys, Ile, D-Ile,Leu, D-Leu, Val, D-Val Phenyl- F D-Phe, Tyr, D-Thr, L-Dopa, His, alanineD-His, Trp, D-Trp, Trans-3, 4, or 5-phenylproline, cis-3, 4, or5-phenylproline Proline P D-Pro, L-1-thioazolidine-4- carboxylic acid,D- or L-1- oxazolidine-4-carboxylic acid Serine S D-Ser, Thr, D-Thr,allo-Thr, Met, D-Met, Met(O), D-Met(O), L-Cys, D-Cys Threonine T D-Thr,Ser, D-Ser, allo-Thr, Met, D-Met, Met(O), D-Met(O), Val, D-Val TyrosineY D-Tyr, Phe, D-Phe, L-Dopa, His, D-His Valine V D-Val, Leu, D-Leu, Ile,D-Ile, Met, D-Met

Aside from the uses described above, such amino acid substitutions mayalso increase protein or peptide stability. The invention encompassesamino acid substitutions that contain, for example, one or morenon-peptide bonds (which replace the peptide bonds) in the protein orpeptide sequence. Also included are substitutions that include aminoacid residues other than naturally occurring L-amino acids, e.g.,D-amino acids or non-naturally occurring or synthetic amino acids, e.g.,β or γ amino acids.

Both identity and similarity can be readily calculated by reference tothe following publications: Computational Molecular Biology, Lesk, A.M., ed., Oxford University Press, New York, 1988; Biocomputing:Informatics and Genome Projects, Smith, D. W., ed., Academic Press, NewYork, 1993; Informatics Computer Analysis of Sequence Data, Part 1,Griffin, A. M., and Griffin, H. G., eds., Humana Press, N.J., 1994;Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press,1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds.,M Stockton Press, New York, 1991.

In addition, the present invention also encompasses substitution ofamino acids based upon the probability of an amino acid substitutionresulting in conservation of function. Such probabilities are determinedby aligning multiple genes with related function and assessing therelative penalty of each substitution to proper gene function. Suchprobabilities are often described in a matrix and are used by somealgorithms (e.g., BLAST, CLUSTALW, GAP, etc.) in calculating percentsimilarity wherein similarity refers to the degree by which one aminoacid may substitute for another amino acid without lose of function. Anexample of such a matrix is the PAM250 or BLOSUM62 matrix.

Aside from the canonical chemically conservative substitutionsreferenced above, the invention also encompasses substitutions which aretypically not classified as conservative, but that may be chemicallyconservative under certain circumstances. Analysis of enzymaticcatalysis for proteases, for example, has shown that certain amino acidswithin the active site of some enzymes may have highly perturbed pKa'sdue to the unique microenvironment of the active site. Such perturbedpKa's could enable some amino acids to substitute for other amino acidswhile conserving enzymatic structure and function. Examples of aminoacids that are known to have amino acids with perturbed pKa's are theGlu-35 residue of Lysozyme, the Ile-16 residue of Chymotrypsin, theHis-159 residue of Papain, etc. The conservation of function relates toeither anomalous protonation or anomalous deprotonation of such aminoacids, relative to their canonical, non-perturbed pKa. The pKaperturbation may enable these amino acids to actively participate ingeneral acid-base catalysis due to the unique ionization environmentwithin the enzyme active site. Thus, substituting an amino acid capableof serving as either a general acid or general base within themicroenvironment of an enzyme active site or cavity, as may be the case,in the same or similar capacity as the wild-type amino acid, wouldeffectively serve as a conservative amino substitution.

Besides conservative amino acid substitution, variants of the presentinvention include, but are not limited to, the following: (i)substitutions with one or more of the non-conserved amino acid residues,where the substituted amino acid residues may or may not be one encodedby the genetic code, or (ii) substitution with one or more of amino acidresidues having a substituent group, or (iii) fusion of the maturepolypeptide with another compound, such as a compound to increase thestability and/or solubility of the polypeptide (for example,polyethylene glycol), or (iv) fusion of the polypeptide with additionalamino acids, such as, for example, an IgG Fc fusion region peptide, orleader or secretory sequence, or a sequence facilitating purification.Such variant polypeptides are deemed to be within the scope of thoseskilled in the art from the teachings herein.

For example, polypeptide variants containing amino acid substitutions ofcharged amino acids with other charged or neutral amino acids mayproduce proteins with improved characteristics, such as lessaggregation. Aggregation of pharmaceutical formulations both reducesactivity and increases clearance due to the aggregate's immunogenicactivity. (Pinckard et al., Clin. Exp. Immnunol. 2:331-340 (1967);Robbins et al., Diabetes 36: 838-845 (1987); Cleland et al., Crit. Rev.Therapeutic Drug Carrier Systems 10:307-377 (1993).)

Moreover, the invention further includes polypeptide variants createdthrough the application of molecular evolution (“DNA Shuffling”)methodology to the polynucleotide disclosed as SEQ ID NO:1 and/or 3, thesequence of the clone submitted in a deposit, and/or the cDNA encodingthe polypeptide disclosed as SEQ ID NO:2 and/or 4. Such DNA Shufflingtechnology is known in the art and more particularly described elsewhereherein (e.g., WPC, Stemmer, PNAS, 91:10747, (1994)), and in the Examplesprovided herein).

Moreover, the invention further includes polypeptide variants createdthrough the application of molecular evolution (“DNA Shuffling”)methodology to the polynucleotide disclosed as SEQ ID NO:3, the sequenceof the clone submitted in a deposit, and/or the cDNA encoding thepolypeptide disclosed as SEQ ID NO:4. Such DNA Shuffling technology isknown in the art and more particularly described elsewhere herein (e.g.,WPC, Stemmer, PNAS, 91:10747, (1994)), and in the Examples providedherein).

A further embodiment of the invention relates to a polypeptide whichcomprises the amino acid sequence of the present invention having anamino acid sequence which contains at least one amino acid substitution,but not more than 50 amino acid substitutions, even more preferably, notmore than 40 amino acid substitutions, still more preferably, not morethan 30 amino acid substitutions, and still even more preferably, notmore than 20 amino acid substitutions. Of course, in order ofever-increasing preference, it is highly preferable for a peptide orpolypeptide to have an amino acid sequence which comprises the aminoacid sequence of the present invention, which contains at least one, butnot more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid substitutions.In specific embodiments, the number of additions, substitutions, and/ordeletions in the amino acid sequence of the present invention orfragments thereof (e.g., the mature form and/or other fragmentsdescribed herein), is 1-5, 5-10, 5-25, 5-50, 10-50 or 50-150,conservative amino acid substitutions are preferable.

Polynucleotide and Polypeptide Fragments

The present invention is directed to polynucleotide fragments of thepolynucleotides of the invention, in addition to polypeptides encodedtherein by said polynucleotides and/or fragments.

In the present invention, a “polynucleotide fragment” refers to a shortpolynucleotide having a nucleic acid sequence which: is a portion ofthat contained in a deposited clone, or encoding the polypeptide encodedby the cDNA in a deposited clone; is a portion of that shown in SEQ IDNO:1 and/or 3 or the complementary strand thereto, or is a portion of apolynucleotide sequence encoding the polypeptide of SEQ ID NO:2 and/or4. The nucleotide fragments of the invention are preferably at leastabout 15 nt, and more preferably at least about 20 nt, still morepreferably at least about 30 nt, and even more preferably, at leastabout 40 nt, at least about 50 nt, at least about 75 nt, or at leastabout 150 nt in length. A fragment “at least 20 nt in length,” forexample, is intended to include 20 or more contiguous bases from thecDNA sequence contained in a deposited clone or the nucleotide sequenceshown in SEQ ID NO:1 and/or 3. In this context “about” includes theparticularly recited value, a value larger or smaller by several (5, 4,3, 2, or 1) nucleotides, at either terminus, or at both termini. Thesenucleotide fragments have uses that include, but are not limited to, asdiagnostic probes and primers as discussed herein. Of course, largerfragments (e.g., 50, 150, 500, 600, 2000 nucleotides) are preferred.

In the present invention, a “polynucleotide fragment” refers to a shortpolynucleotide having a nucleic acid sequence which: is a portion ofthat contained in a deposited clone, or encoding the polypeptide encodedby the cDNA in a deposited clone; is a portion of that shown in SEQ IDNO:3 or the complementary strand thereto, or is a portion of apolynucleotide sequence encoding the polypeptide of SEQ ID NO:4. Thenucleotide fragments of the invention are preferably at least about 15nt, and more preferably at least about 20 nt, still more preferably atleast about 30 nt, and even more preferably, at least about 40 nt, atleast about 50 nt, at least about 75 nt, or at least about 150 nt inlength. A fragment “at least 20 nt in length,” for example, is intendedto include 20 or more contiguous bases from the cDNA sequence containedin a deposited clone or the nucleotide sequence shown in SEQ ID NO:3. Inthis context “about” includes the particularly recited value, a valuelarger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at eitherterminus, or at both termini. These nucleotide fragments have uses thatinclude, but are not limited to, as diagnostic probes and primers asdiscussed herein. Of course, larger fragments (e.g., 50, 150, 500, 600,2000 nucleotides) are preferred.

Moreover, representative examples of polynucleotide fragments of theinvention, include, for example, fragments comprising, or alternativelyconsisting of, a sequence from about nucleotide number 1-50, 51-100,101-150, 151-200, 201-250, 251-300, 301-350, 351-400, 401-450, 451-500,501-550, 551-600, 651-700, 701-750, 751-800, 800-850, 851-900, 901-950,951-1000, 1001-1050, 1051-1100, 1101-1150, 1151-1200, 1201-1250,1251-1300, 1301-1350, 1351-1400, 1401-1450, 1451-1500, 1501-1550,1551-1600, 1601-1650, 1651-1700, 1701-1750, 1751-1800, 1801-1850,1851-1900, 1901-1950, 1951-2000, or 2001 to the end of SEQ ID NO:1and/or 3, or the complementary strand thereto, or the cDNA contained ina deposited clone. In this context “about” includes the particularlyrecited ranges, and ranges larger or smaller by several (5, 4, 3, 2,or 1) nucleotides, at either terminus or at both termini. Preferably,these fragments encode a polypeptide which has biological activity. Morepreferably, these polynucleotides can be used as probes or primers asdiscussed herein. Also encompassed by the present invention arepolynucleotides which hybridize to these nucleic acid molecules understringent hybridization conditions or lower stringency conditions, asare the polypeptides encoded by these polynucleotides.

Moreover, representative examples of polynucleotide fragments of theinvention, include, for example, fragments comprising, or alternativelyconsisting of, a sequence from about nucleotide number 1-50, 51-100,101-150, 151-200, 201-250, 251-300, 301-3.50, 351-400, 401-450, 451-500,501-550, 551-600, 651-700, 701-750, 751-800, 800-850, 851-900, 901-950,951-1000, 1001-1050, 1051-1100, 1101-1150, 1151-1200, 1201-1250,1251-1300, 1301-1350, 1351-1400, 1401-1450, 1451-1500, 1501-1550,1551-1600, 1601-1650, 1651-1700, 1701-1750, 1751-1800, 1801-1850,1851-1900, 1901-1950, 1951-2000, or 2001 to the end of SEQ ID NO:3, orthe complementary strand thereto, or the cDNA contained in a depositedclone. In this context “about” includes the particularly recited ranges,and ranges larger or smaller by several (5, 4, 3, 2, or 1) nucleotides,at either terminus or at both termini. Preferably, these fragmentsencode a polypeptide which has biological activity. More preferably,these polynucleotides can be used as probes or primers as discussedherein. Also encompassed by the present invention are polynucleotideswhich hybridize to these nucleic acid molecules under stringenthybridization conditions or lower stringency conditions, as are thepolypeptides encoded by these polynucleotides.

In the present invention, a “polypeptide fragment” refers to an aminoacid sequence which is a portion of that contained in SEQ ID NO:2 and/or4 or encoded by the cDNA contained in a deposited clone. Protein(polypeptide) fragments may be “free-standing,” or comprised within alarger polypeptide of which the fragment forms a part or region, mostpreferably as a single continuous region. Representative examples ofpolypeptide fragments of the invention, include, for example, fragmentscomprising, or alternatively consisting of, from about amino acid number1-20, 21-40, 41-60, 61-80, 81-100, 102-120, 121-140, 141-160, or 161 tothe end of the coding region. Moreover, polypeptide fragments can beabout 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150amino acids in length. In this context “about” includes the particularlyrecited ranges or values, and ranges or values larger or smaller byseveral (5, 4, 3, 2, or 1) amino acids, at either extreme or at bothextremes. Polynucleotides encoding these polypeptides are alsoencompassed by the invention.

In the present invention, a “polypeptide fragment” refers to an aminoacid sequence which is a portion of that contained in SEQ ID NO:4 orencoded by the cDNA contained in a deposited clone. Protein(polypeptide) fragments may be “free-standing,” or comprised within alarger polypeptide of which the fragment forms a part or region mostpreferably as a single continuous region. Representative examples ofpolypeptide fragments of the invention, include, for example, fragmentscomprising, or alternatively consisting of, from about amino acid number1-20, 21-40, 41-60, 61-80, 81-100, 102-120, 121-140, 141-160, or 161 tothe end of the coding region. Moreover, polypeptide fragments can beabout 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150amino acids in length. In this context “about” includes the particularlyrecited ranges or values, and ranges or values larger or smaller byseveral (5, 4, 3, 2, or 1) amino acids, at either extreme or at bothextremes. Polynucleotides encoding these polypeptides are alsoencompassed by the invention.

Preferred polypeptide fragments include the full-length protein. Furtherpreferred polypeptide fragments include the full-length protein having acontinuous series of deleted residues from the amino or the carboxyterminus, or both. For example, any number of amino acids, ranging from1-60, can be deleted from the amino terminus of the full-lengthpolypeptide. Similarly, any number of amino acids, ranging from 1-30,can be deleted from the carboxy terminus of the full-length protein.Furthermore, any combination of the above amino and carboxy terminusdeletions are preferred. Similarly, polynucleotides encoding thesepolypeptide fragments are also preferred.

Also preferred are polypeptide and polynucleotide fragmentscharacterized by structural or functional domains, such as fragmentsthat comprise alpha-helix and alpha-helix forming regions, beta-sheetand beta-sheet-forming regions, turn and turn-forming regions, coil andcoil-forming regions, hydrophilic regions, hydrophobic regions, alphaamphipathic regions, beta amphipathic regions, flexible regions,surface-forming regions, substrate binding region, and high antigenicindex regions. Polypeptide fragments of SEQ ID NO:2 and/or 4 fallingwithin conserved domains are specifically contemplated by the presentinvention. Moreover, polynucleotides encoding these domains are alsocontemplated.

Other preferred polypeptide fragments are biologically active fragments.Biologically active fragments are those exhibiting activity similar, butnot necessarily identical, to an activity of the polypeptide of thepresent invention. The biological activity of the fragments may includean improved desired activity, or a decreased undesirable activity.Polynucleotides encoding these polypeptide fragments are alsoencompassed by the invention.

In a preferred embodiment, the functional activity displayed by apolypeptide encoded by a polynucleotide fragment of the invention may beone or more biological activities typically associated with thefull-length polypeptide of the invention. Illustrative of thesebiological activities includes the fragments ability to bind to at leastone of the same antibodies which bind to the full-length protein, thefragments ability to interact with at lease one of the same proteinswhich bind to the full-length, the fragments ability to elicit at leastone of the same immune responses as the full-length protein (i.e., tocause the immune system to create antibodies specific to the sameepitope, etc.), the fragments ability to bind to at least one of thesame polynucleotides as the full-length protein, the fragments abilityto bind to a receptor of the full-length protein, the fragments abilityto bind to a ligand of the full-length protein, and the fragmentsability to multimerize with the full-length protein. However, theskilled artisan would appreciate that some fragments may have biologicalactivities which are desirable and directly inapposite to the biologicalactivity of the full-length protein. The functional activity ofpolypeptides of the invention, including fragments, variants,derivatives, and analogs thereof can be determined by numerous methodsavailable to the skilled artisan, some of which are described elsewhereherein.

The present invention encompasses polypeptides comprising, oralternatively consisting of, an epitope of the polypeptide having anamino acid sequence of SEQ ID NO:2 and/or 4, or an epitope of thepolypeptide sequence encoded by a polynucleotide sequence contained inATCC Deposit No:PTA-3434 or encoded by a polynucleotide that hybridizesto the complement of the sequence of SEQ ID NO:1 and/or 3 or containedin ATCC Deposit No:PTA-3434 under stringent hybridization conditions orlower stringency hybridization conditions as defined supra. The presentinvention further encompasses polynucleotide sequences encoding anepitope of a polypeptide sequence of the invention (such as, forexample, the sequence disclosed in SEQ ID NO: 1 and/or 3),polynucleotide sequences of the complementary strand of a polynucleotidesequence encoding an epitope of the invention, and polynucleotidesequences which hybridize to the complementary strand under stringenthybridization conditions or lower stringency hybridization conditionsdefined supra.

The present invention encompasses polypeptides comprising, oralternatively consisting of, an epitope of the polypeptide having anamino acid sequence of SEQ ID NO:4, or an epitope of the polypeptidesequence encoded by a polynucleotide sequence contained in ATCC DepositNo:PTA-3434 or encoded by a polynucleotide that hybridizes to thecomplement of the sequence of SEQ ID NO:3 or contained in ATCC DepositNo:PTA-3434 under stringent hybridization conditions or lower stringencyhybridization conditions as defined supra. The present invention furtherencompasses polynucleotide sequences encoding an epitope of apolypeptide sequence of the invention (such as, for example, thesequence disclosed in SEQ ID NO:1 and/or 3), polynucleotide sequences ofthe complementary strand of a polynucleotide sequence encoding anepitope of the invention, and polynucleotide sequences which hybridizeto the complementary strand under stringent hybridization conditions orlower stringency hybridization conditions defined supra.

The term “epitopes,” as used herein, refers to portions of a polypeptidehaving antigenic or immunogenic activity in an animal, preferably amammal, and most preferably in a human. In a preferred embodiment, thepresent invention encompasses a polypeptide comprising an epitope, aswell as the polynucleotide encoding this polypeptide. An “immunogenicepitope,” as used herein, is defined as a portion of a protein thatelicits an antibody response in an animal, as determined by any methodknown in the art, for example, by the methods for generating antibodiesdescribed infra. (See, for example, Geysen et al., Proc. Natl. Acad.Sci. USA 81:3998-4002 (1983)). The term “antigenic epitope,” as usedherein, is defined as a portion of a protein to which an antibody canimmunospecifically bind its antigen as determined by any method wellknown in the art, for example, by the immunoassays described herein.Immunospecific binding excludes non-specific binding but does notnecessarily exclude cross-reactivity with other antigens. Antigenicepitopes need not necessarily be immunogenic.

Fragments which function as epitopes may be produced by any conventionalmeans. (See, e.g., Houghten, Proc. Natl. Acad. Sci. USA 82:5131-5135(1985), further described in U.S. Pat. No. 4,631,211).

In the present invention, antigenic epitopes preferably contain asequence of at least 4, at least 5, at least 6, at least 7, morepreferably at least 8, at least 9, at least 10, at least 11, at least12, at least 13, at least 14, at least 15, at least 20, at least 25, atleast 30, at least 40, at least 50, and, most preferably, between about15 to about 30 amino acids. Preferred polypeptides comprisingimmunogenic or antigenic epitopes are at least 10, 15, 20, 25, 30, 35,40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acidresidues in length, or longer. Additional non-exclusive preferredantigenic epitopes include the antigenic epitopes disclosed herein, aswell as portions thereof. Antigenic epitopes are useful, for example, toraise antibodies, including monoclonal antibodies, that specificallybind the epitope. Preferred antigenic epitopes include the antigenicepitopes disclosed herein, as well as any combination of two, three,four, five or more of these antigenic epitopes. Antigenic epitopes canbe used as the target molecules in immunoassays. (See, for instance,Wilson et al., Cell 37:767-778 (1984); Sutcliffe et al., Science219:660-666 (1983)).

Similarly, immunogenic epitopes can be used, for example, to induceantibodies according to methods well known in the art. (See, forinstance, Sutcliffe et al., supra; Wilson et al., supra; Chow et al.,Proc. Natl. Acad. Sci. USA 82:910-914; and Bittle et al., J. Gen. Virol.66:2347-2354 (1985). Preferred immunogenic epitopes include theimmunogenic epitopes disclosed herein, as well as any combination oftwo, three, four, five or more of these immunogenic epitopes. Thepolypeptides comprising one or more immunogenic epitopes may bepresented for eliciting an antibody response together with a carrierprotein, such as an albumin, to an animal system (such as rabbit ormouse), or, if the polypeptide is of sufficient length (at least about25 amino acids), the polypeptide may be presented without a carrier.However, immunogenic epitopes comprising as few as 8 to 10 amino acidshave been shown to be sufficient to raise antibodies capable of bindingto, at the very least, linear epitopes in a denatured polypeptide (e.g.,in Western blotting).

Epitope-bearing polypeptides of the present invention may be used toinduce antibodies according to methods well known in the art including,but not limited to, in vivo immunization, in vitro immunization, andphage display methods. See, e.g., Sutcliffe et al., supra; Wilson etal., supra, and Bittle et al., J. Gen. Virol., 66:2347-2354 (1985). Ifin vivo immunization is used, animals may be immunized with freepeptide; however, anti-peptide antibody titer may be boosted by couplingthe peptide to a macromolecular carrier, such as keyhole limpethemacyanin (KLH) or tetanus toxoid. For instance, peptides containingcysteine residues may be coupled to a carrier using a linker such asmaleimidobenzoyl-N-hydroxysuccinimide ester (MBS), while other peptidesmay be coupled to carriers using a more general linking agent such asglutaraldehyde. Animals such as rabbits, rats and mice are immunizedwith either free or carrier-coupled peptides, for instance, byintraperitoneal and/or intradermal injection of emulsions containingabout 100 μg of peptide or carrier protein and Freund's adjuvant or anyother adjuvant known for stimulating an immune response. Several boosterinjections may be needed, for instance, at intervals of about two weeks,to provide a useful titer of anti-peptide antibody which can bedetected, for example, by ELISA assay using free peptide adsorbed to asolid surface. The titer of anti-peptide antibodies in serum from animmunized animal may be increased by selection of anti-peptideantibodies, for instance, by adsorption to the peptide on a solidsupport and elution of the selected antibodies according to methods wellknown in the art.

As one of skill in the art will appreciate, and as discussed above, thepolypeptides of the present invention comprising an immunogenic orantigenic epitope can be fused to other polypeptide sequences. Forexample, the polypeptides of the present invention may be fused with theconstant domain of immunoglobulins (IgA, IgE, IgG, IgM), or portionsthereof (CH1, CH2, CH3, or any combination thereof and portions thereof)resulting in chimeric polypeptides. Such fusion proteins may facilitatepurification and may increase half-life in vivo. This has been shown forchimeric proteins consisting of the first two domains of the humanCD4-polypeptide and various domains of the constant regions of the heavyor light chains of mammalian immunoglobulins. See, e.g., EP 394,827;Traunecker et al., Nature, 331:84-86 (1988). Enhanced delivery of anantigen across the epithelial barrier to the immune system has beendemonstrated for antigens (e.g., insulin) conjugated to an FcRn bindingpartner such as IgG or Fc fragments (see, e.g., PCT Publications WO96/22024 and WO 99/04813). IgG Fusion proteins that have adisulfide-linked dimeric structure due to the IgG portion disulfidebonds have also been found to be more efficient in binding andneutralizing other molecules than monomeric polypeptides or fragmentsthereof alone. See, e.g., Fountoulakis et al., J. Biochem.,270:3958-3964 (1995). Nucleic acids encoding the above epitopes can alsobe recombined with a gene of interest as an epitope tag (e.g., thehemagglutinin (“HA”) tag or flag tag) to aid in detection andpurification of the expressed polypeptide. For example, a systemdescribed by Janknecht et al. allows for the ready purification ofnon-denatured fusion proteins expressed in human cell lines (Janknechtet al., 1991, Proc. Natl. Acad. Sci. USA 88:8972-897). In this system,the gene of interest is subcloned into a vaccinia recombination plasmidsuch that the open reading frame of the gene is translationally fused toan amino-terminal tag consisting of six histidine residues. The tagserves as a matrix binding domain for the fusion protein. Extracts fromcells infected with the recombinant vaccinia virus are loaded onto Ni2+nitriloacetic acid-agarose column and histidine-tagged proteins can beselectively eluted with imidazole-containing buffers.

Additional fusion proteins of the invention may be generated through thetechniques of gene-shuffling, motif-shuffling, exon-shuffling, and/orcodon-shuffling (collectively referred to as “DNA shuffling”). DNAshuffling may be employed to modulate the activities of polypeptides ofthe invention, such methods can be used to generate polypeptides withaltered activity, as well as agonists and antagonists of thepolypeptides. See, generally, U.S. Pat. Nos. 5,605,793; 5,811,238;5,830,721; 5,834,252; and 5,837,458, and Patten et al., Curr. OpinionBiotechnol. 8:724-33 (1997); Harayama, Trends Biotechnol. 16(2):76-82(1998); Hansson, et al., J. Mol. Biol. 287:265-76 (1999); and Lorenzoand Blasco, Biotechniques 24(2):308-13 (1998) (each of these patents andpublications are hereby incorporated by reference in its entirety).

In one embodiment, alteration of polynucleotides corresponding to SEQ IDNO:1 and/or 3 and the polypeptides encoded by these polynucleotides maybe achieved by DNA shuffling. DNA shuffling involves the assembly of twoor more DNA segments by homologous or site-specific recombination togenerate variation in the polynucleotide sequence. In anotherembodiment, polynucleotides of the invention, or the encodedpolypeptides, may be altered by being subjected to random mutagenesis byerror-prone PCR, random nucleotide insertion or other methods prior torecombination. In another embodiment, one or more components, motifs,sections, parts, domains, fragments, etc., of a polynucleotide encodinga polypeptide of the invention may be recombined with one or morecomponents, motifs, sections, parts, domains, fragments, etc. of one ormore heterologous molecules.

In one embodiment, alteration of polynucleotides corresponding to SEQ IDNO:3 and the polypeptides encoded by these polynucleotides may beachieved by DNA shuffling. DNA shuffling involves the assembly of two ormore DNA segments by homologous or site-specific recombination togenerate variation in the polynucleotide sequence. In anotherembodiment, polynucleotides of the invention, or the encodedpolypeptides, may be altered by being subjected to random mutagenesis byerror-prone PCR, random nucleotide insertion or other methods prior torecombination. In another embodiment, one or more components, motifs,sections, parts, domains, fragments, etc., of a polynucleotide encodinga polypeptide of the invention may be recombined with one or morecomponents, motifs, sections, parts, domains, fragments, etc. of one ormore heterologous molecules.

Antibodies

Further polypeptides of the invention relate to antibodies and T-cellantigen receptors (TCR) which immunospecifically bind a polypeptide,polypeptide fragment, or variant of SEQ ID NO:Y, and/or an epitope, ofthe present invention (as determined by immunoassays well known in theart for assaying specific antibody-antigen binding). Antibodies of theinvention include, but are not limited to, polyclonal, monoclonal,monovalent, bispecific, heteroconjugate, multispecific, human, humanizedor chimeric antibodies, single chain antibodies, Fab fragments, F(ab′)fragments, fragments produced by a Fab expression library,anti-idiotypic (anti-Id) antibodies (including, e.g., anti-Id antibodiesto antibodies of the invention), and epitope-binding fragments of any ofthe above. The term “antibody,” as used herein, refers to immunoglobulinmolecules and immunologically active portions of immunoglobulinmolecules, i.e., molecules that contain an antigen binding site thatimmunospecifically binds an antigen. The immunoglobulin molecules of theinvention can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY),class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass ofimmunoglobulin molecule. Moreover, the term “antibody” (Ab) or“monoclonal antibody” (Mab) is meant to include intact molecules, aswell as, antibody fragments (such as, for example, Fab and F(ab′)2fragments) which are capable of specifically binding to protein. Fab andF(ab′)2 fragments lack the Fc fragment of intact antibody, clear morerapidly from the circulation of the animal or plant, and may have lessnon-specific tissue binding than an intact antibody (Wahl et al., J.Nucl. Med. 24:316-325 (1983)). Thus, these fragments are preferred, aswell as the products of a FAB or other immunoglobulin expressionlibrary. Moreover, antibodies of the present invention include chimeric,single chain, and humanized antibodies.

Most preferably the antibodies are human antigen-binding antibodyfragments of the present invention and include, but are not limited to,Fab, Fab′ and F(ab′)2, Fd, single-chain Fvs (scFv), single-chainantibodies, disulfide-linked Fvs (sdFv) and fragments comprising eithera VL or VH domain. Antigen-binding antibody fragments, includingsingle-chain antibodies, may comprise the variable region(s) alone or incombination with the entirety or a portion of the following: hingeregion, CH1, CH2, and CH3 domains. Also included in the invention areantigen-binding fragments also comprising any combination of variableregion(s) with a hinge region, CH1, CH2, and CH3 domains. The antibodiesof the invention may be from any animal origin including birds andmammals. Preferably, the antibodies are human, murine (e.g., mouse andrat), donkey, ship rabbit, goat, guinea pig, camel, horse, or chicken.As used herein, “human” antibodies include antibodies having the aminoacid sequence of a human immunoglobulin and include antibodies isolatedfrom human immunoglobulin libraries or from animals transgenic for oneor more human immunoglobulin and that do not express endogenousimmunoglobulins, as described infra and, for example in, U.S. Pat. No.5,939,598 by Kucherlapati et al.

The antibodies of the present invention may be monospecific, bispecific,trispecific or of greater multispecificity. Multispecific antibodies maybe specific for different epitopes of a polypeptide of the presentinvention or may be specific for both a polypeptide of the presentinvention as well as for a heterologous epitope, such as a heterologouspolypeptide or solid support material. See, e.g., PCT publications WO93/17715; WO 92/08802; WO 91/00360; WO 92/05793; Tutt, et al., J.Immunol. 147:60-69 (1991); U.S. Pat. Nos. 4,474,893; 4,714,681;4,925,648; 5,573,920; 5,601,819; Kostelny et al., J. Immunol.148:1547-1553 (1992).

Antibodies of the present invention may be described or specified interms of the epitope(s) or portion(s) of a polypeptide of the presentinvention which they recognize or specifically bind. The epitope(s) orpolypeptide portion(s) may be specified as described herein, e.g., byN-terminal and C-terminal positions, by size in contiguous amino acidresidues, or listed in the Tables and Figures. Antibodies whichspecifically bind any epitope or polypeptide of the present inventionmay also be excluded. Therefore, the present invention includesantibodies that specifically bind polypeptides of the present invention,and allows for the exclusion of the same.

Antibodies of the present invention may also be described or specifiedin terms of their cross-reactivity. Antibodies that do not bind anyother analog, ortholog, or homologue of a polypeptide of the presentinvention are included. Antibodies that bind polypeptides with at least95%, at least 90%, at least 85%, at least 80%, at least 75%, at least70%, at least 65%, at least 60%, at least 55%, and at least 50% identity(as calculated using methods known in the art and described herein) to apolypeptide of the present invention are also included in the presentinvention. In specific embodiments, antibodies of the present inventioncross-react with murine, rat and/or rabbit homologues of human proteinsand the corresponding epitopes thereof. Antibodies that do not bindpolypeptides with less than 95%, less than 90%, less than 85%, less than80%, less than 75%, less than 70%, less than 65%, less than 60%, lessthan 55%, and less than 50% identity (as calculated using methods knownin the art and described herein) to a polypeptide of the presentinvention are also included in the present invention. In a specificembodiment, the above-described cross-reactivity is with respect to anysingle specific antigenic or immunogenic polypeptide, or combination(s)of 2, 3, 4, 5, or more of the specific antigenic and/or immunogenicpolypeptides disclosed herein. Further included in the present inventionare antibodies which bind polypeptides encoded by polynucleotides whichhybridize to a polynucleotide of the present invention under stringenthybridization conditions (as described herein). Antibodies of thepresent invention may also be described or specified in terms of theirbinding affinity to a polypeptide of the invention. Preferred bindingaffinities include those with a dissociation constant or Kd less than5×10-2 M, 10-2 M, 5×10-3 M, 10-3 M, 5×10-4 M, 10-4 M, 5×10-5 M, 10-5 M,5×10-6 M, 10-6M, 5×10-7 M, 107 M, 5×10-8 M, 10-8 M, 5×10-9 M, 5×10-10 M,10-10 M, 5×10-11 M, 10-11 M, 5×10-12 M, 10-12 M, 5×10-13 M, 5×10-14 M,10-14 M, 5×10-15 M, or 10-15 M.

The invention also provides antibodies that competitively inhibitbinding of an antibody to an epitope of the invention as determined byany method known in the art for determining competitive binding, forexample, the immunoassays described herein. In preferred embodiments,the antibody competitively inhibits binding to the epitope by at least95%, at least 90%, at least 85%, at least 80%, at least 75%, at least70%, at least 60%, or at least 50%.

Antibodies of the present invention may act as agonists or antagonistsof the polypeptides of the present invention. For example, the presentinvention includes antibodies which disrupt the receptor/ligandinteractions with the polypeptides of the invention either partially orfully. Preferably, antibodies of the present invention bind an antigenicepitope disclosed herein, or a portion thereof. The invention featuresboth receptor-specific antibodies and ligand-specific antibodies. Theinvention also features receptor-specific antibodies which do notprevent ligand binding but prevent receptor activation. Receptoractivation (i.e., signaling) may be determined by techniques describedherein or otherwise known in the art. For example, receptor activationcan be determined by detecting the phosphorylation (e.g., tyrosine orserine/threonine) of the receptor or its substrate byimmunoprecipitation followed by western blot analysis (for example, asdescribed supra). In specific embodiments, antibodies are provided thatinhibit ligand activity or receptor activity by at least 95%, at least90%, at least 85%, at least 80%, at least 75%, at least 70%, at least60%, or at least 50% of the activity in absence of the antibody.

The invention also features receptor-specific antibodies which bothprevent ligand binding and receptor activation as well as antibodiesthat recognize the receptor-ligand complex, and, preferably, do notspecifically recognize the unbound receptor or the unbound ligand.Likewise, included in the invention are neutralizing antibodies whichbind the ligand and prevent binding of the ligand to the receptor, aswell as antibodies which bind the ligand, thereby preventing receptoractivation, but do not prevent the ligand from binding the receptor.Further included in the invention are antibodies which activate thereceptor. These antibodies may act as receptor agonists, i.e.,potentiate or activate either all or a subset of the biologicalactivities of the ligand-mediated receptor activation, for example, byinducing dimerization of the receptor. The antibodies may be specifiedas agonists, antagonists or inverse agonists for biological activitiescomprising the specific biological activities of the peptides of theinvention disclosed herein. The above antibody agonists can be madeusing methods known in the art. See, e.g., PCT publication WO 96/40281;U.S. Pat. No. 5,811,097; Deng et al., Blood 92(6):1981-1988 (1998); Chenet al., Cancer Res. 58(16):3668-3678 (1998); Harrop et al., J. Immunol.161(4):1786-1794 (1998); Zhu et al., Cancer Res. 58(15):3209-3214(1998); Yoon et al., J. Immunol. 160(7):3170-3179 (1998); Prat et al.,J. Cell. Sci. 111(Pt2):237-247 (1998); Pitard et al., J. Immunol.Methods 205(2):177-190 (1997); Liautard et al., Cytokine 9(4):233-241(1997); Carlson et al., J. Biol. Chem. 272(17):11295-11301 (1997);Taryman et al., Neuron 14(4):755-762 (1995); Muller et al., Structure6(9):1153-1167 (1998); Bartunek et al., Cytokine 8(1):14-20 (1996)(which are all incorporated by reference herein in their entireties).

Antibodies of the present invention may be used, for example, but notlimited to, to purify, detect, and target the polypeptides of thepresent invention, including both in vitro and in vivo diagnostic andtherapeutic methods. For example, the antibodies have use inimmunoassays for qualitatively and quantitatively measuring levels ofthe polypeptides of the present invention in biological samples. See,e.g., Harlow et al., Antibodies: A Laboratory Manual, (Cold SpringHarbor Laboratory Press, 2nd ed. 1988) (incorporated by reference hereinin its entirety).

As discussed in more detail below, the antibodies of the presentinvention may be used either alone or in combination with othercompositions. The antibodies may further be recombinantly fused to aheterologous polypeptide at the N— or C-terminus or chemicallyconjugated (including covalently and non-covalently conjugations) topolypeptides or other compositions. For example, antibodies of thepresent invention may be recombinantly fused or conjugated to moleculesuseful as labels in detection assays and effector molecules such asheterologous polypeptides, drugs, radionucleotides, or toxins. See,e.g., PCT publications WO 92/08495; WO 91/14438; WO 89/12624; U.S. Pat.No. 5,314,995; and EP 396,387.

The antibodies of the invention include derivatives that are modified,i.e., by the covalent attachment of any type of molecule to the antibodysuch that covalent attachment does not prevent the antibody fromgenerating an anti-idiotypic response. For example, but not by way oflimitation, the antibody derivatives include antibodies that have beenmodified, e.g., by glycosylation, acetylation, pegylation,phosphorylation, amidation, derivatization by known protecting/blockinggroups, proteolytic cleavage, linkage to a cellular ligand or otherprotein, etc. Any of numerous chemical modifications may be carried outby known techniques, including, but not limited to specific chemicalcleavage, acetylation, formylation, metabolic synthesis of tunicamycin,etc. Additionally, the derivative may contain one or more non-classicalamino acids.

The antibodies of the present invention may be generated by any suitablemethod known in the art.

The antibodies of the present invention may comprise polyclonalantibodies. Methods of preparing polyclonal antibodies are known to theskilled artisan (Harlow, et al., Antibodies: A Laboratory Manual, (Coldspring Harbor Laboratory Press, 2^(nd) ed. (1988); and CurrentProtocols, Chapter 2; which are hereby incorporated herein by referencein its entirety). In a preferred method, a preparation of the HLLRCR-1protein is prepared and purified to render it substantially free ofnatural contaminants. Such a preparation is then introduced into ananimal in order to produce polyclonal antisera of greater specificactivity. For example, a polypeptide of the invention can beadministered to various host animals including, but not limited to,rabbits, mice, rats, etc. to induce the production of sera containingpolyclonal antibodies specific for the antigen. The administration ofthe polypeptides of the present invention may entail one or moreinjections of an immunizing agent and, if desired, an adjuvant. Variousadjuvants may be used to increase the immunological response, dependingon the host species, and include but are not limited to, Freund's(complete and incomplete), mineral gels such as aluminum hydroxide,surface active substances such as lysolecithin, pluronic polyols,polyanions, peptides, oil emulsions, keyhole limpet hemocyanins,dinitrophenol, and potentially useful human adjuvants such as BCG(bacille Calmette-Guerin) and corynebacterium parvum. Such adjuvants arealso well known in the art. For the purposes of the invention,“immunizing agent” may be defined as a polypeptide of the invention,including fragments, variants, and/or derivatives thereof, in additionto fusions with heterologous polypeptides and other forms of thepolypeptides described herein.

Typically, the immunizing agent and/or adjuvant will be injected in themammal by multiple subcutaneous or intraperitoneal injections, thoughthey may also be given intramuscularly, and/or through IV). Theimmunizing agent may include polypeptides of the present invention or afusion protein or variants thereof. Depending upon the nature of thepolypeptides (i.e., percent hydrophobicity, percent hydrophilicity,stability, net charge, isoelectric point etc.), it may be useful toconjugate the immunizing agent to a protein known to be immunogenic inthe mammal being immunized. Such conjugation includes either chemicalconjugation by derivitizing active chemical functional groups to boththe polypeptide of the present invention and the immunogenic proteinsuch that a covalent bond is formed, or through fusion-protein basedmethodology, or other methods known to the skilled artisan. Examples ofsuch immunogenic proteins include, but are not limited to keyhole limpethemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsininhibitor. Various adjuvants may be used to increase the immunologicalresponse, depending on the host species, including but not limited toFreund's (complete and incomplete), mineral gels such as aluminumhydroxide, surface active substances such as lysolecithin, pluronicpolyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin,dinitrophenol, and potentially useful human adjuvants such as BCG(bacille Calmette-Guerin) and Corynebacterium parvum. Additionalexamples of adjuvants which may be employed includes the MPL-TDMadjuvant (monophosphoryl lipid A, synthetic trehalose dicorynomycolate).The immunization protocol may be selected by one skilled in the artwithout undue experimentation.

The antibodies of the present invention may comprise monoclonalantibodies. Monoclonal antibodies may be prepared using hybridomamethods, such as those described by Kohler and Milstein, Nature, 256:495(1975) and U.S. Pat. No. 4,376,110, by Harlow, et al., Antibodies: ALaboratory Manual, (Cold spring Harbor Laboratory Press, 2^(nd) ed.(1988), by Hammerling, et al., Monoclonal Antibodies and T-CellHybridomas (Elsevier, N.Y., pp. 563-681 (1981); Köhler et al., Eur. J.Immunol. 6:511 (1976); Köhler et al., Eur. J. Immunol. 6:292 (1976), orother methods known to the artisan. Other examples of methods which maybe employed for producing monoclonal antibodies includes, but are notlimited to, the human B-cell hybridoma technique (Kosbor et al., 1983,Immunology Today 4:72; Cole et al., 1983, Proc. Natl. Acad. Sci. USA80:2026-2030), and the EBV-hybridoma technique (Cole et al., 1985,Monoclonal Antibodies And Cancer Therapy, Alan R. Liss, Inc., pp.77-96). Such antibodies may be of any immunoglobulin class includingIgG, IgM, IgE, IgA, IgD and any subclass thereof. The hybridomaproducing the mAb of this invention may be cultivated in vitro or invivo. Production of high titers of mAbs in vivo makes this the presentlypreferred method of production.

In a hybridoma method, a mouse, a humanized mouse, a mouse with a humanimmune system, hamster, or other appropriate host animal, is typicallyimmunized with an immunizing agent to elicit lymphocytes that produce orare capable of producing antibodies that will specifically bind to theimmunizing agent. Alternatively, the lymphocytes may be immunized invitro.

The immunizing agent will typically include polypeptides of the presentinvention or a fusion protein thereof. Preferably, the immunizing agentconsists of an HLLRCR-1 polypeptide or, more preferably, with a HLLRCR-1polypeptide-expressing cell. Such cells may be cultured in any suitabletissue culture medium; however, it is preferable to culture cells inEarle's modified Eagle's medium supplemented with 10% fetal bovine serum(inactivated at about 56 degrees C.), and supplemented with about 10 g/lof nonessential amino acids, about 1,000 U/ml of penicillin, and about100 ug/ml of streptomycin. Generally, either peripheral bloodlymphocytes (“PBLs”) are used if cells of human origin are desired, orspleen cells or lymph node cells are used if non-human mammalian sourcesare desired. The lymphocytes are then fused with an immortalized cellline using a suitable fusing agent, such as polyethylene glycol, to forma hybridoma cell (Goding, Monoclonal Antibodies: Principles andPractice, Academic Press, (1986), pp. 59-103). Immortalized cell linesare usually transformed mammalian cells, particularly myeloma cells ofrodent, bovine and human origin. Usually, rat or mouse myeloma celllines are employed. The hybridoma cells may be cultured in a suitableculture medium that preferably contains one or more substances thatinhibit the growth or survival of the unfused, immortalized cells. Forexample, if the parental cells lack the enzyme hypoxanthine guaninephosphoribosyl transferase (HGPRT or HPRT), the culture medium for thehybridomas typically will include hypoxanthine, aminopterin, andthymidine (“HAT medium”), which substances prevent the growth ofHGPRT-deficient cells.

Preferred immortalized cell lines are those that fuse efficiently,support stable high level expression of antibody by the selectedantibody-producing cells, and are sensitive to a medium such as HATmedium. More preferred immortalized cell lines are murine myeloma lines,which can be obtained, for instance, from the Salk Institute CellDistribution Center, San Diego, Calif. and the American Type CultureCollection, Manassas, Va. More preferred are the parent myeloma cellline (SP2O) as provided by the ATCC. As inferred throughout thespecification, human myeloma and mouse-human heteromyeloma cell linesalso have been described for the production of human monoclonalantibodies (Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al.,Monoclonal Antibody Production Techniques and Applications, MarcelDekker, Inc., New York, (1987) pp. 51-63).

The culture medium in which the hybridoma cells are cultured can then beassayed for the presence of monoclonal antibodies directed against thepolypeptides of the present invention. Preferably, the bindingspecificity of monoclonal antibodies produced by the hybridoma cells isdetermined by immunoprecipitation or by an in vitro binding assay, suchas radioimmunoassay (RIA) or enzyme-linked immunoabsorbant assay(ELISA). Such techniques are known in the art and within the skill ofthe artisan. The binding affinity of the monoclonal antibody can, forexample, be determined by the Scatchard analysis of Munson and Pollart,Anal. Biochem., 107:220 (1980).

After the desired hybridoma cells are identified, the clones may besubcloned by limiting dilution procedures and grown by standard methods(Goding, supra, and/or according to Wands et al. (Gastroenterology80:225-232 (1981)). Suitable culture media for this purpose include, forexample, Dulbecco's Modified Eagle's Medium and RPMI-1640.Alternatively, the hybridoma cells may be grown in vivo as ascites in amammal.

The monoclonal antibodies secreted by the subclones may be isolated orpurified from the culture medium or ascites fluid by conventionalimmunoglobulin purification procedures such as, for example, proteinA-sepharose, hydroxyapatite chromatography, gel exclusionchromatography, gel electrophoresis, dialysis, or affinitychromatography.

The skilled artisan would acknowledge that a variety of methods exist inthe art for the production of monoclonal antibodies and thus, theinvention is not limited to their sole production in hydridomas. Forexample, the monoclonal antibodies may be made by recombinant DNAmethods, such as those described in U.S. Pat. No. 4,816,567. In thiscontext, the term “monoclonal antibody” refers to an antibody derivedfrom a single eukaryotic, phage, or prokaryotic clone. The DNA encodingthe monoclonal antibodies of the invention can be readily isolated andsequenced using conventional procedures (e.g., by using oligonucleotideprobes that are capable of binding specifically to genes encoding theheavy and light chains of murine antibodies, or such chains from human,humanized, or other sources). The hydridoma cells of the invention serveas a preferred source of such DNA. Once isolated, the DNA may be placedinto expression vectors, which are then transformed into host cells suchas Simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cellsthat do not otherwise produce immunoglobulin protein, to obtain thesynthesis of monoclonal antibodies in the recombinant host cells. TheDNA also may be modified, for example, by substituting the codingsequence for human heavy and light chain constant domains in place ofthe homologous murine sequences (U.S. Pat. No. 4,816,567; Morrison etal, supra) or by covalently joining to the immunoglobulin codingsequence all or part of the coding sequence for a non-immunoglobulinpolypeptide. Such a non-immunoglobulin polypeptide can be substitutedfor the constant domains of an antibody of the invention, or can besubstituted for the variable domains of one antigen-combining site of anantibody of the invention to create a chimeric bivalent antibody.

The antibodies may be monovalent antibodies. Methods for preparingmonovalent antibodies are well known in the art. For example, one methodinvolves recombinant expression of immunoglobulin light chain andmodified heavy chain. The heavy chain is truncated generally at anypoint in the Fc region so as to prevent heavy chain crosslinking.Alternatively, the relevant cysteine residues are substituted withanother amino acid residue or are deleted so as to prevent crosslinking.

In vitro methods are also suitable for preparing monovalent antibodies.Digestion of antibodies to produce fragments thereof, particularly, Fabfragments, can be accomplished using routine techniques known in theart. Monoclonal antibodies can be prepared using a wide variety oftechniques known in the art including the use of hybridoma, recombinant,and phage display technologies, or a combination thereof. For example,monoclonal antibodies can be produced using hybridoma techniquesincluding those known in the art and taught, for example, in Harlow etal., Antibodies: A Laboratory Manual, (Cold Spring Harbor LaboratoryPress, 2nd ed. 1988); Hammerling, et al., in: Monoclonal Antibodies andT-Cell Hybridomas 563-681 (Elsevier, N.Y., 1981) (said referencesincorporated by reference in their entireties). The term “monoclonalantibody” as used herein is not limited to antibodies produced throughhybridoma technology. The term “monoclonal antibody” refers to anantibody that is derived from a single clone, including any eukaryotic,prokaryotic, or phage clone, and not the method by which it is produced.

Methods for producing and screening for specific antibodies usinghybridoma technology are routine and well known in the art and arediscussed in detail in the Examples described herein. In a non-limitingexample, mice can be immunized with a polypeptide of the invention or acell expressing such peptide. Once an immune response is detected, e.g.,antibodies specific for the antigen are detected in the mouse serum, themouse spleen is harvested and splenocytes isolated. The splenocytes arethen fused by well known techniques to any suitable myeloma cells, forexample cells from cell line SP20 available from the ATCC. Hybridomasare selected and cloned by limited dilution. The hybridoma clones arethen assayed by methods known in the art for cells that secreteantibodies capable of binding a polypeptide of the invention. Ascitesfluid, which generally contains high levels of antibodies, can begenerated by immunizing mice with positive hybridoma clones.

Accordingly, the present invention provides methods of generatingmonoclonal antibodies as well as antibodies produced by the methodcomprising culturing a hybridoma cell secreting an antibody of theinvention wherein, preferably, the hybridoma is generated by fusingsplenocytes isolated from a mouse immunized with an antigen of theinvention with myeloma cells and then screening the hybridomas resultingfrom the fusion for hybridoma clones that secrete an antibody able tobind a polypeptide of the invention.

Antibody fragments which recognize specific epitopes may be generated byknown techniques. For example, Fab and F(ab′)2 fragments of theinvention may be produced by proteolytic cleavage of immunoglobulinmolecules, using enzymes such as papain (to produce Fab fragments) orpepsin (to produce F(ab′)2 fragments). F(ab′)2 fragments contain thevariable region, the light chain constant region and the CH1 domain ofthe heavy chain.

For example, the antibodies of the present invention can also begenerated using various phage display methods known in the art. In phagedisplay methods, functional antibody domains are displayed on thesurface of phage particles which carry the polynucleotide sequencesencoding them. In a particular embodiment, such phage can be utilized todisplay antigen binding domains expressed from a repertoire orcombinatorial antibody library (e.g., human or murine). Phage expressingan antigen binding domain that binds the antigen of interest can beselected or identified with antigen, e.g., using labeled antigen orantigen bound or captured to a solid surface or bead. Phage used inthese methods are typically filamentous phage including fd and M13binding domains expressed from phage with Fab, Fv or disulfidestabilized Fv antibody domains recombinantly fused to either the phagegene III or gene VIII protein. Examples of phage display methods thatcan be used to make the antibodies of the present invention includethose disclosed in Brinkman et al., J. Immunol. Methods 182:41-50(1995); Ames et al., J. Immunol. Methods 184:177-186 (1995);Kettleborough et al., Eur. J. Immnunol. 24:952-958 (1994); Persic etal., Gene 187 9-18 (1997); Burton et al., Advances in Immunology57:191-280 (1994); PCT application No. PCT/GB91/01134; PCT publicationsWO 90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO95/15982; WO 95/20401; and U.S. Pat. Nos. 5,698,426; 5,223,409;5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698;5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743 and 5,969,108;each of which is incorporated herein by reference in its entirety.

As described in the above references, after phage selection, theantibody coding regions from the phage can be isolated and used togenerate whole antibodies, including human antibodies, or any otherdesired antigen binding fragment, and expressed in any desired host,including mammalian cells, insect cells, plant cells, yeast, andbacteria, e.g., as described in detail below. For example, techniques torecombinantly produce Fab, Fab′ and F(ab′)2 fragments can also beemployed using methods known in the art such as those disclosed in PCTpublication WO 92/22324; Mullinax et al., BioTechniques 12(6):864-869(1992); and Sawai et al., AJRI 34:26-34 (1995); and Better et al.,Science 240:1041-1043 (1988) (said references incorporated by referencein their entireties). Examples of techniques which can be used toproduce single-chain Fvs and antibodies include those described in U.S.Pat. Nos. 4,946,778 and 5,258,498; Huston et al., Methods in Enzymology203:46-88 (1991); Shu et al., PNAS 90:7995-7999 (1993); and Skerra etal., Science 240:1038-1040 (1988).

For some uses, including in vivo use of antibodies in humans and invitro detection assays, it may be preferable to use chimeric, humanized,or human antibodies. A chimeric antibody is a molecule in whichdifferent portions of the antibody are derived from different animalspecies, such as antibodies having a variable region derived from amurine monoclonal antibody and a human immunoglobulin constant region.Methods for producing chimeric antibodies are known in the art. Seee.g., Morrison, Science 229:1202 (1985); Oi et al., BioTechniques 4:214(1986); Gillies et al., (1989) J. Immunol. Methods 125:191-202; Cabillyet al., Taniguchi et al., EP 171496; Morrison et al., EP 173494;Neuberger et al., WO 8601533; Robinson et al., WO 8702671; Boulianne etal., Nature 312:643 (1984); Neuberger et al., Nature 314:268 (1985);U.S. Pat. Nos. 5,807,715; 4,816,567; and 4,816397, which areincorporated herein by reference in their entirety. Humanized antibodiesare antibody molecules from non-human species antibody that binds thedesired antigen having one or more complementarity determining regions(CDRs) from the non-human species and a framework regions from a humanimmunoglobulin molecule. Often, framework residues in the humanframework regions will be substituted with the corresponding residuefrom the CDR donor antibody to alter, preferably improve, antigenbinding. These framework substitutions are identified by methods wellknown in the art, e.g., by modeling of the interactions of the CDR andframework residues to identify framework residues important for antigenbinding and sequence comparison to identify unusual framework residuesat particular positions. (See, e.g., Queen et al., U.S. Pat. No.5,585,089; Riechmann et al., Nature 332:323 (1988), which areincorporated herein by reference in their entireties.) Antibodies can behumanized using a variety of techniques known in the art including, forexample, CDR-grafting (EP 239,400; PCT publication WO 91/09967; U.S.Pat. Nos. 5,225,539; 5,530,101; and 5,585,089), veneering or resurfacing(EP 592,106; EP 519,596; Padlan, Molecular Immunology 28(4/5):489-498(1991); Studnicka et al., Protein Engineering 7(6):805-814 (1994);Roguska. et al., PNAS 91:969-973 (1994)), and chain shuffling (U.S. Pat.No. 5,565,332). Generally, a humanized antibody has one or more aminoacid residues introduced into it from a source that is non-human. Thesenon-human amino acid residues are often referred to as “import”residues, which are typically taken from an “import” variable domain.Humanization can be essentially performed following the methods ofWinter and co-workers (Jones et al., Nature, 321:522-525 (1986);Reichmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science,239:1534-1536 (1988), by substituting rodent CDRs or CDR sequences forthe corresponding sequences of a human antibody. Accordingly, such“humanized” antibodies are chimeric antibodies (U.S. Pat. No.4,816,567), wherein substantially less than an intact human variabledomain has been substituted by the corresponding sequence from anon-human species. In practice, humanized antibodies are typically humanantibodies in which some CDR residues and possible some FR residues aresubstituted from analogous sites in rodent antibodies.

In general, the humanized antibody will comprise substantially all of atleast one, and typically two, variable domains, in which all orsubstantially all of the CDR regions correspond to those of a non-humanimmunoglobulin and all or substantially all of the FR regions are thoseof a human immunoglobulin consensus sequence. The humanized antibodyoptimally also will comprise at least a portion of an immunoglobulinconstant region (Fc), typically that of a human immunoglobulin (Jones etal., Nature, 321:522-525 (1986); Riechmann et al., Nature 332:323-329(1988)1 and Presta, Curr. Op. Struct. Biol., 2:593-596 (1992).

Completely human antibodies are particularly desirable for therapeutictreatment of human patients. Human antibodies can be made by a varietyof methods known in the art including phage display methods describedabove using antibody libraries derived from human immunoglobulinsequences. See also, U.S. Pat. Nos. 4,444,887 and 4,716,111; and PCTpublications WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO96/34096, WO 96/33735, and WO 91/10741; each of which is incorporatedherein by reference in its entirety. The techniques of cole et al., andBoerder et al., are also available for the preparation of humanmonoclonal antibodies (cole et al., Monoclonal Antibodies and CancerTherapy, Alan R. Riss, (1985); and Boerner et al., J. Immunol.,147(l):86-95, (1991)).

Human antibodies can also be produced using transgenic mice which areincapable of expressing functional endogenous immunoglobulins, but whichcan express human immunoglobulin genes. For example, the human heavy andlight chain immunoglobulin gene complexes may be introduced randomly orby homologous recombination into mouse embryonic stem cells.Alternatively, the human variable region, constant region, and diversityregion may be introduced into mouse embryonic stem cells in addition tothe human heavy and light chain genes. The mouse heavy and light chainimmunoglobulin genes may be rendered non-functional separately orsimultaneously with the introduction of human immunoglobulin loci byhomologous recombination. In particular, homozygous deletion of the JHregion prevents endogenous antibody production. The modified embryonicstem cells are expanded and microinjected into blastocysts to producechimeric mice. The chimeric mice are then bred to produce homozygousoffspring which express human antibodies. The transgenic mice areimmunized in the normal fashion with a selected antigen, e.g., all or aportion of a polypeptide of the invention. Monoclonal antibodiesdirected against the antigen can be obtained from the immunized,transgenic mice using conventional hybridoma technology. The humanimmunoglobulin transgenes harbored by the transgenic mice rearrangeduring B cell differentiation, and subsequently undergo class switchingand somatic mutation. Thus, using such a technique, it is possible toproduce therapeutically useful IgG, IgA, IgM and IgE antibodies. For anoverview of this technology for producing human antibodies, see Lonbergand Huszar, Int. Rev. Immunol. 13:65-93 (1995). For a detaileddiscussion of this technology for producing human antibodies and humanmonoclonal antibodies and protocols for producing such antibodies, see,e.g., PCT publications WO 98/24893; WO 92/01047; WO 96/34096; WO96/33735; European Patent No. 0 598 877; U.S. Pat. Nos. 5,413,923;5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318;5,885,793; 5,916,771; and 5,939,598, which are incorporated by referenceherein in their entirety. In addition, companies such as Abgenix, Inc.(Freemont, Calif.), Genpharm (San Jose, Calif.), and Medarex, Inc.(Princeton, N.J.) can be engaged to provide human antibodies directedagainst a selected antigen using technology similar to that describedabove.

Similarly, human antibodies can be made by introducing humanimmunoglobulin loci into transgenic animals, e.g., mice in which theendogenous immunoglobulin genes have been partially or completelyinactivated. Upon challenge, human antibody production is observed,which closely resembles that seen in humans in all respects, includinggene rearrangement, assembly, and creation of an antibody repertoire.This approach is described, for example, in U.S. Pat. Nos. 5,545,807;5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,106, and in thefollowing scientific publications: Marks et al., Biotechnol., 10:779-783(1992); Lonberg et al., Nature 368:856-859 (1994); Fishwild et al.,Nature Biotechnol., 14:845-51 (1996); Neuberger, Nature Biotechnol.,14:826 (1996); Lonberg and Huszer, Intern. Rev. Immunol., 13:65-93(1995).

Completely human antibodies which recognize a selected epitope can begenerated using a technique referred to as “guided selection.” In thisapproach a selected non-human monoclonal antibody, e.g., a mouseantibody, is used to guide the selection of a completely human antibodyrecognizing the same epitope. (Jespers et al., Bio/technology 12:899-903(1988)).

Further, antibodies to the polypeptides of the invention can, in turn,be utilized to generate anti-idiotype antibodies that “mimic”polypeptides of the invention using techniques well known to thoseskilled in the art. (See, e.g., Greenspan & Bona, FASEB J. 7(5):437-444;(1989) and Nissinoff, J. Immunol. 147(8):2429-2438 (1991)). For example,antibodies which bind to and competitively inhibit polypeptidemultimerization and/or binding of a polypeptide of the invention to aligand can be used to generate anti-idiotypes that “mimic” thepolypeptide multimerization and/or binding domain and, as a consequence,bind to and neutralize polypeptide and/or its ligand. Such neutralizinganti-idiotypes or Fab fragments of such anti-idiotypes can be used intherapeutic regimens to neutralize polypeptide ligand. For example, suchanti-idiotypic antibodies can be used to bind a polypeptide of theinvention and/or to bind its ligands/receptors, and thereby block itsbiological activity.

Such anti-idiotypic antibodies capable of binding to the HLLRCR-1polypeptide can be produced in a two-step procedure. Such a method makesuse of the fact that antibodies are themselves antigens, and therefore,it is possible to obtain an antibody that binds to a second antibody. Inaccordance with this method, protein specific antibodies are used toimmunize an animal, preferably a mouse. The splenocytes of such ananimal are then used to produce hybridoma cells, and the hybridoma cellsare screened to identify clones that produce an antibody whose abilityto bind to the protein-specific antibody can be blocked by thepolypeptide. Such antibodies comprise anti-idiotypic antibodies to theprotein-specific antibody and can be used to immunize an animal toinduce formation of further protein-specific antibodies.

The antibodies of the present invention may be bispecific antibodies.Bispecific antibodies are monoclonal, Preferably human or humanized,antibodies that have binding specificities for at least two differentantigens. In the present invention, one of the binding specificities maybe directed towards a polypeptide of the present invention, the othermay be for any other antigen, and preferably for a cell-surface protein,receptor, receptor subunit, tissue-specific antigen, virally derivedprotein, virally encoded envelope protein, bacterially derived protein,or bacterial surface protein, etc.

Methods for making bispecific antibodies are known in the art.Traditionally, the recombinant production of bispecific antibodies isbased on the co-expression of two immunoglobulin heavy-chain/light-chainpairs, where the two heavy chains have different specificities (Milsteinand Cuello, Nature, 305:537-539 (1983). Because of the random assortmentof immunoglobulin heavy and light chains, these hybridomas (quadromas)produce a potential mixture of ten different antibody molecules, ofwhich only one has the correct bispecific structure. The purification ofthe correct molecule is usually accomplished by affinity chromatographysteps. Similar procedures are disclosed in WO 93/08829, published 13 May1993, and in Traunecker et al., EMBO J., 10:3655-3659 (1991).

Antibody variable domains with the desired binding specificities(antibody-antigen combining sites) can be fused to immunoglobulinconstant domain sequences. The fusion preferably is with animmunoglobulin heavy-chain constant domain, comprising at least part ofthe hinge, CH2, and CH3 regions. It is preferred to have the firstheavy-chain constant region (CH1) containing the site necessary forlight-chain binding present in at least one of the fusions. DNAsencoding the immunoglobulin heavy-chain fusions and, if desired, theimmunoglobulin light chain, are inserted into separate expressionvectors, and are co-transformed into a suitable host organism. Forfurther details of generating bispecific antibodies see, for exampleSuresh et al., Meth. In Enzym., 121:210 (1986).

Heteroconjugate antibodies are also contemplated by the presentinvention. Heteroconjugate antibodies are composed of two covalentlyjoined antibodies. Such antibodies have, for example, been proposed totarget immune system cells to unwanted cells (U.S. Pat. No. 4,676,980),and for the treatment of HIV infection (WO 91/00360; WO 92/20373; andEP03089). It is contemplated that the antibodies may be prepared invitro using known methods in synthetic protein chemistry, includingthose involving crosslinking agents. For example, immunotoxins may beconstructed using a disulfide exchange reaction or by forming athioester bond. Examples of suitable reagents for this purpose includeiminothiolate and methyl-4-mercaptobutyrimidate and those disclosed, forexample, in U.S. Pat. No. 4,676,980.

Polynucleotides Encoding Antibodies

The invention further provides polynucleotides comprising a nucleotidesequence encoding an antibody of the invention and fragments thereof.The invention also encompasses polynucleotides that hybridize understringent or lower stringency hybridization conditions, e.g., as definedsupra, to polynucleotides that encode an antibody, preferably, thatspecifically binds to a polypeptide of the invention, preferably, anantibody that binds to a polypeptide having the amino acid sequence ofSEQ ID NO:Y.

The polynucleotides may be obtained, and the nucleotide sequence of thepolynucleotides determined, by any method known in the art. For example,if the nucleotide sequence of the antibody is known, a polynucleotideencoding the antibody may be assembled from chemically synthesizedoligonucleotides (e.g., as described in Kutmeier et al., BioTechniques17:242 (1994)), which, briefly, involves the synthesis of overlappingoligonucleotides containing portions of the sequence encoding theantibody, annealing and ligating of those oligonucleotides, and thenamplification of the ligated oligonucleotides by PCR.

Alternatively, a polynucleotide encoding an antibody may be generatedfrom nucleic acid from a suitable source. If a clone containing anucleic acid encoding a particular antibody is not available, but thesequence of the antibody molecule is known, a nucleic acid encoding theimmunoglobulin may be chemically synthesized or obtained from a suitablesource (e.g., an antibody cDNA library, or a cDNA library generatedfrom, or nucleic acid, preferably poly A+ RNA, isolated from, any tissueor cells expressing the antibody, such as hybridoma cells selected toexpress an antibody of the invention) by PCR amplification usingsynthetic primers hybridizable to the 3′ and 5′ ends of the sequence orby cloning using an oligonucleotide probe specific for the particulargene sequence to identify, e.g., a cDNA clone from a cDNA library thatencodes the antibody. Amplified nucleic acids generated by PCR may thenbe cloned into replicable cloning vectors using any method well known inthe art.

Once the nucleotide sequence and corresponding amino acid sequence ofthe antibody is determined, the nucleotide sequence of the antibody maybe manipulated using methods well known in the art for the manipulationof nucleotide sequences, e.g., recombinant DNA techniques, site directedmutagenesis, PCR, etc. (see, for example, the techniques described inSambrook et al., 1990, Molecular Cloning, A Laboratory Manual, 2d Ed.,Cold Spring Harbor Laboratory, Cold Spring Harbor, NY and Ausubel etal., eds., 1998, Current Protocols in Molecular Biology, John Wiley &Sons, NY, which are both incorporated by reference herein in theirentireties ), to generate antibodies having a different amino acidsequence, for example to create amino acid substitutions, deletions,and/or insertions.

In a specific embodiment, the amino acid sequence of the heavy and/orlight chain variable domains may be inspected to identify the sequencesof the complementarity determining regions (CDRs) by methods that arewell know in the art, e.g., by comparison to known amino acid sequencesof other heavy and light chain variable regions to determine the regionsof sequence hypervariability. Using routine recombinant DNA techniques,one or more of the CDRs may be inserted within framework regions, e.g.,into human framework regions to humanize a non-human antibody, asdescribed supra. The framework regions may be naturally occurring orconsensus framework regions, and preferably human framework regions(see, e.g., Chothia et al., J. Mol. Biol. 278: 457-479 (1998) for alisting of human framework regions). Preferably, the polynucleotidegenerated by the combination of the framework regions and CDRs encodesan antibody that specifically binds a polypeptide of the invention.Preferably, as discussed supra, one or more amino acid substitutions maybe made within the framework regions, and, preferably, the amino acidsubstitutions improve binding of the antibody to its antigen.Additionally, such methods may be used to make amino acid substitutionsor deletions of one or more variable region cysteine residuesparticipating in an intrachain disulfide bond to generate antibodymolecules lacking one or more intrachain disulfide bonds. Otheralterations to the polynucleotide are encompassed by the presentinvention and within the skill of the art.

In addition, techniques developed for the production of “chimericantibodies” (Morrison et al., Proc. Natl. Acad. Sci. 81:851-855 (1984);Neuberger et al., Nature 312:604-608 (1984); Takeda et al., Nature314:452-454 (1985)) by splicing genes from a mouse antibody molecule ofappropriate antigen specificity together with genes from a humanantibody molecule of appropriate biological activity can be used. Asdescribed supra, a chimeric antibody is a molecule in which differentportions are derived from different animal species, such as those havinga variable region derived from a murine mAb and a human immunoglobulinconstant region, e.g., humanized antibodies.

Alternatively, techniques described for the production of single chainantibodies (U.S. Pat. No. 4,946,778; Bird, Science 242:423- 42 (1988);Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); and Wardet al., Nature 334:544-54 (1989)) can be adapted to produce single chainantibodies. Single chain antibodies are formed by linking the heavy andlight chain fragments of the Fv region via an amino acid bridge,resulting in a single chain polypeptide. Techniques for the assembly offunctional Fv fragments in E. coli may also be used (Skerra et al.,Science 242:1038- 1041 (1988)).

More preferably, a clone encoding an antibody of the present inventionmay be obtained according to the method described in the Example sectionherein.

Methods of Producing Antibodies

The antibodies of the invention can be produced by any method known inthe art for the synthesis of antibodies, in particular, by chemicalsynthesis or preferably, by recombinant expression techniques.

Recombinant expression of an antibody of the invention, or fragment,derivative or analog thereof, (e.g., a heavy or light chain of anantibody of the invention or a single chain antibody of the invention),requires construction of an expression vector containing apolynucleotide that encodes the antibody. Once a polynucleotide encodingan antibody molecule or a heavy or light chain of an antibody, orportion thereof (preferably containing the heavy or light chain variabledomain), of the invention has been obtained, the vector for theproduction of the antibody molecule may be produced by recombinant DNAtechnology using techniques well known in the art. Thus, methods forpreparing a protein by expressing a polynucleotide containing anantibody encoding nucleotide sequence are described herein. Methodswhich are well known to those skilled in the art can be used toconstruct expression vectors containing antibody coding sequences andappropriate transcriptional and translational control signals. Thesemethods include, for example, in vitro recombinant DNA techniques,synthetic techniques, and in vivo genetic recombination. The invention,thus, provides replicable vectors comprising a nucleotide sequenceencoding an antibody molecule of the invention, or a heavy or lightchain thereof, or a heavy or light chain variable domain, operablylinked to a promoter. Such vectors may include the nucleotide sequenceencoding the constant region of the antibody molecule (see, e.g., PCTPublication WO 86/05807; PCT Publication WO 89/01036; and U.S. Pat. No.5,122,464) and the variable domain of the antibody may be cloned intosuch a vector for expression of the entire heavy or light chain.

The expression vector is transferred to a host cell by conventionaltechniques and the transfected cells are then cultured by conventionaltechniques to produce an antibody of the invention. Thus, the inventionincludes host cells containing a polynucleotide encoding an antibody ofthe invention, or a heavy or light chain thereof, or a single chainantibody of the invention, operably linked to a heterologous promoter.In preferred embodiments for the expression of double-chainedantibodies, vectors encoding both the heavy and light chains may beco-expressed in the host cell for expression of the entireimmunoglobulin molecule, as detailed below.

A variety of host-expression vector systems may be utilized to expressthe antibody molecules of the invention. Such host-expression systemsrepresent vehicles by which the coding sequences of interest may beproduced and subsequently purified, but also represent cells which may,when transformed or transfected with the appropriate nucleotide codingsequences, express an antibody molecule of the invention in situ. Theseinclude but are not limited to microorganisms such as bacteria (e.g., E.coli, B. subtilis) transformed with recombinant bacteriophage DNA,plasmid DNA or cosmid DNA expression vectors containing antibody codingsequences; yeast (e.g., Saccharomyces, Pichia) transformed withrecombinant yeast expression vectors containing antibody codingsequences; insect cell systems infected with recombinant virusexpression vectors (e.g., baculovirus) containing antibody codingsequences; plant cell systems infected with recombinant virus expressionvectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus,TMV) or transformed with recombinant plasmid expression vectors (e.g.,Ti plasmid) containing antibody coding sequences; or mammalian cellsystems (e.g., COS, CHO, BHK, 293, 3T3 cells) harboring recombinantexpression constructs containing promoters derived from the genome ofmammalian cells (e.g., metallothionein promoter) or from mammalianviruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5Kpromoter). Preferably, bacterial cells such as Escherichia coli, andmore preferably, eukaryotic cells, especially for the expression ofwhole recombinant antibody molecule, are used for the expression of arecombinant antibody molecule. For example, mammalian cells such asChinese hamster ovary cells (CHO), in conjunction with a vector such asthe major intermediate early gene promoter element from humancytomegalovirus is an effective expression system for antibodies(Foecking et al., Gene 45:101 (1986); Cockett et al., Bio/Technology 8:2(1990)).

In bacterial systems, a number of expression vectors may beadvantageously selected depending upon the use intended for the antibodymolecule being expressed. For example, when a large quantity of such aprotein is to be produced, for the generation of pharmaceuticalcompositions of an antibody molecule, vectors which direct theexpression of high levels of fusion protein products that are readilypurified may be desirable. Such vectors include, but are not limited, tothe E. coli expression vector pUR278 (Ruther et al., EMBO J. 2:1791(1983)), in which the antibody coding sequence may be ligatedindividually into the vector in frame with the lac Z coding region sothat a fusion protein is produced; pIN vectors (Inouye & Inouye, NucleicAcids Res. 13:3101-3109 (1985); Van Heeke & Schuster, J. Biol. Chem.24:5503-5509 (1989)); and the like. pGEX vectors may also be used toexpress foreign polypeptides as fusion proteins with glutathioneS-transferase (GST). In general, such fusion proteins are soluble andcan easily be purified from lysed cells by adsorption and binding tomatrix glutathione-agarose beads followed by elution in the presence offree glutathione. The pGEX vectors are designed to include thrombin orfactor Xa protease cleavage sites so that the cloned target gene productcan be released from the GST moiety.

In an insect system, Autographa californica nuclear polyhedrosis virus(AcNPV) is used as a vector to express foreign genes. The virus grows inSpodoptera frugiperda cells. The antibody coding sequence may be clonedindividually into non-essential regions (for example the polyhedringene) of the virus and placed under control of an AcNPV promoter (forexample the polyhedrin promoter).

In mammalian host cells, a number of viral-based expression systems maybe utilized. In cases where an adenovirus is used as an expressionvector, the antibody coding sequence of interest may be ligated to anadenovirus transcription/translation control complex, e.g., the latepromoter and tripartite leader sequence. This chimeric gene may then beinserted in the adenovirus genome by in vitro or in vivo recombination.Insertion in a non- essential region of the viral genome (e.g., regionE1 or E3) will result in a recombinant virus that is viable and capableof expressing the antibody molecule in infected hosts. (e.g., see Logan& Shenk, Proc. Natl. Acad. Sci. USA 81:355-359 (1984)). Specificinitiation signals may also be required for efficient translation ofinserted antibody coding sequences. These signals include the ATGinitiation codon and adjacent sequences. Furthermore, the initiationcodon must be in phase with the reading frame of the desired codingsequence to ensure translation of the entire insert. These exogenoustranslational control signals and initiation codons can be of a varietyof origins, both natural and synthetic. The efficiency of expression maybe enhanced by the inclusion of appropriate transcription enhancerelements, transcription terminators, etc. (see Bittner et al., Methodsin Enzymol. 153:51-544 (1987)).

In addition, a host cell strain may be chosen which modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Such modifications (e.g.,glycosylation) and processing (e.g., cleavage) of protein products maybe important for the function of the protein. Different host cells havecharacteristic and specific mechanisms for the post-translationalprocessing and modification of proteins and gene products. Appropriatecell lines or host systems can be chosen to ensure the correctmodification and processing of the foreign protein expressed. To thisend, eukaryotic host cells which possess the cellular machinery forproper processing of the primary transcript, glycosylation, andphosphorylation of the gene product may be used. Such mammalian hostcells include but are not limited to CHO, VERY, BHK, Hela, COS, MDCK,293, 3T3, WI38, and in particular, breast cancer cell lines such as, forexample, BT483, Hs578T, HTB2, BT20 and T47D, and normal mammary glandcell line such as, for example, CRL7030 and Hs578Bst.

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. For example, cell lines which stably expressthe antibody molecule may be engineered. Rather than using expressionvectors which contain viral origins of replication, host cells can betransformed with DNA controlled by appropriate expression controlelements (e.g., promoter, enhancer, sequences, transcriptionterminators, polyadenylation sites, etc.), and a selectable marker.Following the introduction of the foreign DNA, engineered cells may beallowed to grow for 1-2 days in an enriched media, and then are switchedto a selective media. The selectable marker in the recombinant plasmidconfers resistance to the selection and allows cells to stably integratethe plasmid into their chromosomes and grow to form foci which in turncan be cloned and expanded into cell lines. This method mayadvantageously be used to engineer cell lines which express the antibodymolecule. Such engineered cell lines may be particularly useful inscreening and evaluation of compounds that interact directly orindirectly with the antibody molecule.

A number of selection systems may be used, including but not limited tothe herpes simplex virus thymidine kinase (Wigler et al., Cell 11:223(1977)), hypoxanthine-guanine phosphoribosyltransferase (Szybalska &Szybalski, Proc. Natl. Acad. Sci. USA 48:202 (1992)), and adeninephosphoribosyltransferase (Lowy et al., Cell 22:817 (1980)) genes can beemployed in tk-, hgprt- or aprt-cells, respectively. Also,antimetabolite resistance can be used as the basis of selection for thefollowing genes: dhfr, which confers resistance to methotrexate (Wigleret al., Natl. Acad. Sci. USA 77:357 (1980); O'Hare et al., Proc. Natl.Acad. Sci. USA 78:1527 (1981)); gpt, which confers resistance tomycophenolic acid (Mulligan & Berg, Proc. Natl. Acad. Sci. USA 78:2072(1981)); neo, which confers resistance to the aminoglycoside G-418Clinical Pharmacy 12:488-505; Wu and Wu, Biotherapy 3:87-95 (1991);Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32:573-596 (1993); Mulligan,Science 260:926-932 (1993); and Morgan and Anderson, Ann. Rev. Biochem.62:191-217 (1993); May, 1993, TIB TECH 11(5):155-215); and hygro, whichconfers resistance to hygromycin (Santerre et al., Gene 30:147 (1984)).Methods commonly known in the art of recombinant DNA technology may beroutinely applied to select the desired recombinant clone, and suchmethods are described, for example, in Ausubel et al. (eds.), CurrentProtocols in Molecular Biology, John Wiley & Sons, NY (1993); Kriegler,Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY(1990); and in Chapters 12 and 13, Dracopoli et al. (eds), CurrentProtocols in Human Genetics, John Wiley & Sons, NY (1994);Colberre-Garapin et al., J. Mol. Biol. 150:1 (1981), which areincorporated by reference herein in their entireties.

The expression levels of an antibody molecule can be increased by vectoramplification (for a review, see Bebbington and Hentschel, The use ofvectors based on gene amplification for the expression of cloned genesin mammalian cells in DNA cloning, Vol.3. (Academic Press, New York,1987)). When a marker in the vector system expressing antibody isamplifiable, increase in the level of inhibitor present in culture ofhost cell will increase the number of copies of the marker gene. Sincethe amplified region is associated with the antibody gene, production ofthe antibody will also increase (Crouse et al., Mol. Cell. Biol. 3:257(1983)).

The host cell may be co-transfected with two expression vectors of theinvention, the first vector encoding a heavy chain derived polypeptideand the second vector encoding a light chain derived polypeptide. Thetwo vectors may contain identical selectable markers which enable equalexpression of heavy and light chain polypeptides. Alternatively, asingle vector may be used which encodes, and is capable of expressing,both heavy and light chain polypeptides. In such situations, the lightchain should be placed before the heavy chain to avoid an excess oftoxic free heavy chain (Proudfoot, Nature 322:52 (1986); Kohler, Proc.Natl. Acad. Sci. USA 77:2197 (1980)). The coding sequences for the heavyand light chains may comprise cDNA or genomic DNA.

Once an antibody molecule of the invention has been produced by ananimal, chemically synthesized, or recombinantly expressed, it may bepurified by any method known in the art for purification of animmunoglobulin molecule, for example, by chromatography (e.g., ionexchange, affinity, particularly by affinity for the specific antigenafter Protein A, and sizing column chromatography), centrifugation,differential solubility, or by any other standard technique for thepurification of proteins. In addition, the antibodies of the presentinvention or fragments thereof can be fused to heterologous polypeptidesequences described herein or otherwise known in the art, to facilitatepurification.

The present invention encompasses antibodies recombinantly fused orchemically conjugated (including both covalently and non-covalentlyconjugations) to a polypeptide (or portion thereof, preferably at least10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 amino acids of thepolypeptide) of the present invention to generate fusion proteins. Thefusion does not necessarily need to be direct, but may occur throughlinker sequences. The antibodies may be specific for antigens other thanpolypeptides (or portion thereof, preferably at least 10, 20, 30, 40,50, 60, 70, 80, 90 or 100 amino acids of the polypeptide) of the presentinvention. For example, antibodies may be used to target thepolypeptides of the present invention to particular cell types, eitherin vitro or in vivo, by fusing or conjugating the polypeptides of thepresent invention to antibodies specific for particular cell surfacereceptors. Antibodies fused or conjugated to the polypeptides of thepresent invention may also be used in in vitro immunoassays andpurification methods using methods known in the art. See e.g., Harbor etal., supra, and PCT publication WO 93/21232; EP 439,095; Naramura etal., Immunol. Lett. 39:91-99 (1994); U.S. Pat. No. 5,474,981; Gillies etal., PNAS 89:1428-1432 (1992); Fell et al., J. Immunol.146:2446-2452(1991), which are incorporated by reference in theirentireties.

The present invention further includes compositions comprising thepolypeptides of the present invention fused or conjugated to antibodydomains other than the variable regions. For example, the polypeptidesof the present invention may be fused or conjugated to an antibody Fcregion, or portion thereof. The antibody portion fused to a polypeptideof the present invention may comprise the constant region, hinge region,CH1 domain, CH2 domain, and CH3 domain or any combination of wholedomains or portions thereof. The polypeptides may also be fused orconjugated to the above antibody portions to form multimers. Forexample, Fc portions fused to the polypeptides of the present inventioncan form dimers through disulfide bonding between the Fc portions.Higher multimeric forms can be made by fusing the polypeptides toportions of IgA and IgM. Methods for fusing or conjugating thepolypeptides of the present invention to antibody portions are known inthe art. See, e.g., U.S. Pat. Nos. 5,336,603; 5,622,929; 5,359,046;5,349,053; 5,447,851; 5,112,946; EP 307,434; EP 367,166; PCTpublications WO 96/04388; WO 91/06570; Ashkenazi et al., Proc. Natl.Acad. Sci. USA 88:10535-10539 (1991); Zheng et al., J. Immunol.154:5590-5600 (1995); and Vil et al., Proc. Natl. Acad. Sci. USA89:11337-11341(1992) (said references incorporated by reference in theirentireties).

As discussed, supra, the polypeptides corresponding to a polypeptide,polypeptide fragment, or a variant of SEQ ID NO:Y may be fused orconjugated to the above antibody portions to increase the in vivo halflife of the polypeptides or for use in immunoassays using methods knownin the art. Further, the polypeptides corresponding to SEQ ID NO:Y maybe fused or conjugated to the above antibody portions to facilitatepurification. One reported example describes chimeric proteinsconsisting of the first two domains of the human CD4-polypeptide andvarious domains of the constant regions of the heavy or light chains ofmammalian immunoglobulins. (EP 394,827; Traunecker et al., Nature331:84-86 (1988). The polypeptides of the present invention fused orconjugated to an antibody having disulfide- linked dimeric structures(due to the IgG) may also be more efficient in binding and neutralizingother molecules, than the monomeric secreted protein or protein fragmentalone. (Fountoulakis et al., J. Biochem. 270:3958-3964 (1995)). In manycases, the Fc part in a fusion protein is beneficial in therapy anddiagnosis, and thus can result in, for example, improved pharmacokineticproperties. (EP A 232,262). Alternatively, deleting the Fc part afterthe fusion protein has been expressed, detected, and purified, would bedesired. For example, the Fc portion may hinder therapy and diagnosis ifthe fusion protein is used as an antigen for immunizations. In drugdiscovery, for example, human proteins, such as hIL-5, have been fusedwith Fc portions for the purpose of high-throughput screening assays toidentify antagonists of hIL-5. (See, Bennett et al., J. MolecularRecognition 8:52-58 (1995); Johanson et al., J. Biol. Chem.270:9459-9471 (1995).

Moreover, the antibodies or fragments thereof of the present inventioncan be fused to marker sequences, such as a peptide to facilitatepurification. In preferred embodiments, the marker amino acid sequenceis a hexa-histidine peptide, such as the tag provided in a pQE vector(QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311), amongothers, many of which are commercially available. As described in Gentzet al., Proc. Natl. Acad. Sci. USA 86:821-824 (1989), for instance,hexa-histidine provides for convenient purification of the fusionprotein. Other peptide tags useful for purification include, but are notlimited to, the “HA” tag, which corresponds to an epitope derived fromthe influenza hemagglutinin protein (Wilson et al., Cell 37:767 (1984))and the “flag” tag.

The present invention further encompasses antibodies or fragmentsthereof conjugated to a diagnostic or therapeutic agent. The antibodiescan be used diagnostically to, for example, monitor the development orprogression of a tumor as part of a clinical testing procedure to, e.g.,determine the efficacy of a given treatment regimen. Detection can befacilitated by coupling the antibody to a detectable substance. Examplesof detectable substances include various enzymes, prosthetic groups,fluorescent materials, luminescent materials, bioluminescent materials,radioactive materials, positron emitting metals using various positronemission tomographies, and nonradioactive paramagnetic metal ions. Thedetectable substance may be coupled or conjugated either directly to theantibody (or fragment thereof) or indirectly, through an intermediate(such as, for example, a linker known in the art) using techniques knownin the art. See, for example, U.S. Pat. No. 4,741,900 for metal ionswhich can be conjugated to antibodies for use as diagnostics accordingto the present invention. Examples of suitable enzymes includehorseradish peroxidase, alkaline phosphatase, beta-galactosidase, oracetylcholinesterase; examples of suitable prosthetic group complexesinclude streptavidin/biotin and avidin/biotin; examples of suitablefluorescent materials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; an example of a luminescent material includesluminol; examples of bioluminescent materials include luciferase,luciferin, and aequorin; and examples of suitable radioactive materialinclude 125I, 131I, 111In or 99Tc.

Further, an antibody or fragment thereof may be conjugated to atherapeutic moiety such as a cytotoxin, e.g., a cytostatic or cytocidalagent, a therapeutic agent or a radioactive metal ion, e.g.,alpha-emitters such as, for example, 213Bi. A cytotoxin or cytotoxicagent includes any agent that is detrimental to cells. Examples includepaclitaxol, cytochalasin B, gramicidin D, ethidium bromide, emetine,mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin,doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone,mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids,procaine, tetracaine, lidocaine, propranolol, and puromycin and analogsor homologues thereof. Therapeutic agents include, but are not limitedto, antimetabolites (e.g., methotrexate, 6-mercaptopurine,6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylatingagents (e.g., mechlorethamine, thioepa chlorambucil, melphalan,carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan,dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamineplatinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin(formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin(formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)),and anti-mitotic agents (e.g., vincristine and vinblastine).

The conjugates of the invention can be used for modifying a givenbiological response, the therapeutic agent or drug moiety is not to beconstrued as limited to classical chemical therapeutic agents. Forexample, the drug moiety may be a protein or polypeptide possessing adesired biological activity. Such proteins may include, for example, atoxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin;a protein such as tumor necrosis factor, a-interferon, β-interferon,nerve growth factor, platelet derived growth factor, tissue plasminogenactivator, an apoptotic agent, e.g., TNF-alpha, TNF-beta, AIM I (See,International Publication No. WO 97/33899), AIM II (See, InternationalPublication No. WO 97/34911), Fas Ligand (Takahashi et al., Int.Immunol., 6:1567-1574 (1994)), VEGI (See, International Publication No.WO 99/23105), a thrombotic agent or an anti-angiogenic agent, e.g.,angiostatin or endostatin; or, biological response modifiers such as,for example, lymphokines, interleukin-1 (“IL-1”), interleukin-2(“IL-2”), interleukin-6 (“IL-6”), granulocyte macrophage colonystimulating factor (“GM-CSF”), granulocyte colony stimulating factor(“G-CSF”), or other growth factors.

Antibodies may also be attached to solid supports, which areparticularly useful for immunoassays or purification of the targetantigen. Such solid supports include, but are not limited to, glass,cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride orpolypropylene.

Techniques for conjugating such therapeutic moiety to antibodies arewell known, see, e.g., Arnon et al., “Monoclonal Antibodies ForImmunotargeting Of Drugs In Cancer Therapy”, in Monoclonal AntibodiesAnd Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss,Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, inControlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53(Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of CytotoxicAgents In Cancer Therapy: A Review”, in Monoclonal Antibodies '84:Biological And Clinical Applications, Pinchera et al. (eds.), pp.475-506 (1985); “Analysis, Results, And Future Prospective Of TheTherapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, inMonoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al.(eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., “ThePreparation And Cytotoxic Properties Of Antibody-Toxin Conjugates”,immunol. Rev. 62:119-58 (1982).

Alternatively, an antibody can be conjugated to a second antibody toform an antibody heteroconjugate as described by Segal in U.S. Pat. No.4,676,980, which is incorporated herein by reference in its entirety.

An antibody, with or without a therapeutic moiety conjugated to it,administered alone or in combination with cytotoxic factor(s) and/orcytokine(s) can be used as a therapeutic.

The present invention also encompasses the creation of syntheticantibodies directed against the polypeptides of the present invention.One example of synthetic antibodies is described in Radrizzani, M., etal., Medicina, (Aires), 59(6):753-8, (1999)). Recently, a new class ofsynthetic antibodies has been described and are referred to asmolecularly imprinted polymers (MIPs) (Semorex, Inc.). Antibodies,peptides, and enzymes are often used as molecular recognition elementsin chemical and biological sensors. However, their lack of stability andsignal transduction mechanisms limits their use as sensing devices.Molecularly imprinted polymers (MIPs) are capable of mimicking thefunction of biological receptors but with less stability constraints.Such polymers provide high sensitivity and selectivity while maintainingexcellent thermal and mechanical stability. MIPs have the ability tobind to small molecules and to target molecules such as organics andproteins' with equal or greater potency than that of natural antibodies.These “super” MIPs have higher affinities for their target and thusrequire lower concentrations for efficacious binding.

During synthesis, the MIPs are imprinted so as to have complementarysize, shape, charge and functional groups of the selected target byusing the target molecule itself (such as a polypeptide, antibody,etc.), or a substance having a very similar structure, as its “print” or“template.” MIPs can be derivatized with the same reagents afforded toantibodies. For example, fluorescent ‘super’ MIPs can be coated ontobeads or wells for use in highly sensitive separations or assays, or foruse in high throughput screening of proteins.

Moreover, MIPs based upon the structure of the polypeptide(s) of thepresent invention may be useful in screening for compounds that bind tothe polypeptide(s) of the invention. Such a MIP would serve the role ofa synthetic “receptor” by minimicking the native architecture of thepolypeptide. In fact, the ability of a MIP to serve the role of asynthetic receptor has already been demonstrated for the estrogenreceptor (Ye, L., Yu, Y., Mosbach, K, Analyst., 126(6):760-5, (2001);Dickert, F, L., Hayden, O., Halikias, K, P, Analyst., 126(6):766-71,(2001)). A synthetic receptor may either be mimicked in its entirety(e.g., as the entire protein), or mimicked as a series of short peptidescorresponding to the protein (Rachkov, A., Minoura, N. Biochim, Biophys,Acta., 1544(1-2):255-66, (2001)). Such a synthetic receptor MIPs may beemployed in any one or more of the screening methods described elsewhereherein.

MIPs have also been shown to be useful in “sensing” the presence of itsmimicked molecule (Cheng, Z., Wang, E., Yang, X, Biosens, Bioelectron.,16(3):179-85, (2001); Jenkins, A, L., Yin, R., Jensen, J. L, Analyst.,126(6):798-802, (2001); Jenkins, A, L., Yin, R., Jensen, J. L, Analyst.,126(6):798-802, (2001)). For example, a MIP designed using a polypeptideof the present invention may be used in assays designed to identify, andpotentially quantitate, the level of said polypeptide in a sample. Sucha MIP may be used as a substitute for any component described in theassays, or kits, provided herein (e.g., ELISA, etc.).

A number of methods may be employed to create MIPs to a specificreceptor, ligand, polypeptide, peptide, organic molecule. Severalpreferred methods are described by Esteban et al in J. Anal, Chem.,370(7):795-802, (2001), which is hereby incorporated herein by referencein its entirety in addition to any references cited therein. Additionalmethods are known in the art and are encompassed by the presentinvention, such as for example, Hart, B, R., Shea, K, J. J. Am. Chem,Soc., 123(9):2072-3, (2001); and Quaglia, M., Chenon, K., Hall, A, J.,De, Lorenzi, E., Sellergren, B, J. Am. Chem, Soc., 123(10):2146-54,(2001); which are hereby incorporated by reference in their entiretyherein.

Uses for Antibodies Directed Against Polypeptides of the Invention

The antibodies of the present invention have various utilities. Forexample, such antibodies may be used in diagnostic assays to detect thepresence or quantification of the polypeptides of the invention in asample. Such a diagnostic assay may be comprised of at least two steps.The first, subjecting a sample with the antibody, wherein the sample isa tissue (e.g., human, animal, etc.), biological fluid (e.g., blood,urine, sputum, semen, amniotic fluid, saliva, etc.), biological extract(e.g., tissue or cellular homogenate, etc.), a protein microchip (e.g.,See Arenkov P, et al., Anal Biochem., 278(2):123-131 (2000)), or achromatography column, etc. And a second step involving thequantification of antibody bound to the substrate. Alternatively, themethod may additionally involve a first step of attaching the antibody,either covalently, electrostatically, or reversibly, to a solid support,and a second step of subjecting the bound antibody to the sample, asdefined above and elsewhere herein.

Various diagnostic assay techniques are known in the art, such ascompetitive binding assays, direct or indirect sandwich assays andimmunoprecipitation assays conducted in either heterogeneous orhomogenous phases (Zola, Monoclonal Antibodies: A Manual of Techniques,CRC Press, Inc., (1987), pp 147-158). The antibodies used in thediagnostic assays can be labeled with a detectable moiety. Thedetectable moiety should be capable of producing, either directly orindirectly, a detectable signal. For example, the detectable moiety maybe a radioisotope, such as 2H, 14C, 32P, or 125I, a florescent orchemiluminescent compound, such as fluorescein isothiocyanate,rhodamine, or luciferin, or an enzyme, such as alkaline phosphatase,beta-galactosidase, green fluorescent protein, or horseradishperoxidase. Any method known in the art for conjugating the antibody tothe detectable moiety may be employed, including those methods describedby Hunter et al., Nature, 144:945 (1962); Dafvid et al., Biochem.,13:1014 (1974); Pain et al., J. Immunol. Metho., 40:219(1981); andNygren, J. Histochem. And Cytochem., 30:407 (1982).

Antibodies directed against the polypeptides of the present inventionare useful for the affinity purification of such polypeptides fromrecombinant cell culture or natural sources. In this process, theantibodies against a particular polypeptide are immobilized on asuitable support, such as a Sephadex resin or filter paper, usingmethods well known in the art. The immobilized antibody then iscontacted with a sample containing the polypeptides to be purified, andthereafter the support is washed with a suitable solvent that willremove substantially all the material in the sample except for thedesired polypeptides, which are bound to the immobilized antibody.Finally, the support is washed with another suitable solvent that willrelease the desired polypeptide from the antibody.

Immunophenotyping

The antibodies of the invention may be utilized for immunophenotyping ofcell lines and biological samples. The translation product of the geneof the present invention may be useful as a cell specific marker, ormore specifically as a cellular marker that is differentially expressedat various stages of differentiation and/or maturation of particularcell types. Monoclonal antibodies directed against a specific epitope,or combination of epitopes, will allow for the screening of cellularpopulations expressing the marker. Various techniques can be utilizedusing monoclonal antibodies to screen for cellular populationsexpressing the marker(s), and include magnetic separation usingantibody-coated magnetic beads, “panning” with antibody attached to asolid matrix (i.e., plate), and flow cytometry (See, e.g., U.S. Pat. No.5,985,660; and Morrison et al., Cell, 96:737-49 (1999)).

These techniques allow for the screening of particular populations ofcells, such as might be found with hematological malignancies (i.e.minimal residual disease (MRD) in acute leukemic patients) and“non-self” cells in transplantations to prevent Graft-versus-HostDisease (GVHD). Alternatively, these techniques allow for the screeningof hematopoietic stem and progenitor cells capable of undergoingproliferation and/or differentiation, as might be found in humanumbilical cord blood.

Assays For Antibody Binding

The antibodies of the invention may be assayed for immunospecificbinding by any method known in the art. The immunoassays which can beused include but are not limited to competitive and non-competitiveassay systems using techniques such as western blots, radioimmunoassays,ELISA (enzyme linked immunosorbent assay), “sandwich” immunoassays,immunoprecipitation assays, precipitin reactions, gel diffusionprecipitin reactions, immunodiffusion assays, agglutination assays,complement-fixation assays, immunoradiometric assays, fluorescentimmunoassays, protein A immunoassays, to name but a few. Such assays areroutine and well known in the art (see, e.g., Ausubel et al, eds, 1994,Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc.,New York, which is incorporated by reference herein in its entirety).Exemplary immunoassays are described briefly below (but are not intendedby way of limitation).

Immunoprecipitation protocols generally comprise lysing a population ofcells in a lysis buffer such as RIPA buffer (1% NP-40 or Triton X-100,1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 0.01 M sodium phosphateat pH 7.2, 1% Trasylol) supplemented with protein phosphatase and/orprotease inhibitors (e.g., EDTA, PMSF, aprotinin, sodium vanadate),adding the antibody of interest to the cell lysate, incubating for aperiod of time (e.g., 1-4 hours) at 4° C., adding protein A and/orprotein G sepharose beads to the cell lysate, incubating for about anhour or more at 4° C., washing the beads in lysis buffer andresuspending the beads in SDS/sample buffer. The ability of the antibodyof interest to immunoprecipitate a particular antigen can be assessedby, e.g., western blot analysis. One of skill in the art would beknowledgeable as to the parameters that can be modified to increase thebinding of the antibody to an antigen and decrease the background (e.g.,pre-clearing the cell lysate with sepharose beads). For furtherdiscussion regarding immunoprecipitation protocols see, e.g., Ausubel etal, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, JohnWiley & Sons, Inc., New York at 10.16.1.

Western blot analysis generally comprises preparing protein samples,electrophoresis of the protein samples in a polyacrylamide gel (e.g.,8%-20% SDS-PAGE depending on the molecular weight of the antigen),transferring the protein sample from the polyacrylamide gel to amembrane such as nitrocellulose, PVDF or nylon, blocking the membrane inblocking solution (e.g., PBS with 3% BSA or non-fat milk), washing themembrane in washing buffer (e.g., PBS-Tween 20), blocking the membranewith primary antibody (the antibody of interest) diluted in blockingbuffer, washing the membrane in washing buffer, blocking the membranewith a secondary antibody (which recognizes the primary antibody, e.g.,an anti-human antibody) conjugated to an enzymatic substrate (e.g.,horseradish peroxidase or alkaline phosphatase) or radioactive molecule(e.g., 32P or 125I) diluted in blocking buffer, washing the membrane inwash buffer, and detecting the presence of the antigen. One of skill inthe art would be knowledgeable as to the parameters that can be modifiedto increase the signal detected and to reduce the background noise. Forfurther discussion regarding western blot protocols see, e.g., Ausubelet al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, JohnWiley & Sons, Inc., New York at 10.8.1.

ELISAs comprise preparing antigen, coating the well of a 96 wellmicrotiter plate with the antigen, adding the antibody of interestconjugated to a detectable compound such as an enzymatic substrate(e.g., horseradish peroxidase or alkaline phosphatase) to the well andincubating for a period of time, and detecting the presence of theantigen. In ELISAs the antibody of interest does not have to beconjugated to a detectable compound; instead, a second antibody (whichrecognizes the antibody of interest) conjugated to a detectable compoundmay be added to the well. Further, instead of coating the well with theantigen, the antibody may be coated to the well. In this case, a secondantibody conjugated to a detectable compound may be added following theaddition of the antigen of interest to the coated well. One of skill inthe art would be knowledgeable as to the parameters that can be modifiedto increase the signal detected as well as other variations of ELISAsknown in the art. For further discussion regarding ELISAs see, e.g.,Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol.1, John Wiley & Sons, Inc., New York at 11.2.1.

The binding affinity of an antibody to an antigen and the off-rate of anantibody-antigen interaction can be determined by competitive bindingassays. One example of a competitive binding assay is a radioimmunoassaycomprising the incubation of labeled antigen (e.g., 3H or 125I) with theantibody of interest in the presence of increasing amounts of unlabeledantigen, and the detection of the antibody bound to the labeled antigen.The affinity of the antibody of interest for a particular antigen andthe binding off-rates can be determined from the data by scatchard plotanalysis. Competition with a second antibody can also be determinedusing radioimmunoassays. In this case, the antigen is incubated withantibody of interest conjugated to a labeled compound (e.g., 3H or 125I)in the presence of increasing amounts of an unlabeled second antibody.

Therapeutic Uses Of Antibodies

The present invention is further directed to antibody-based therapieswhich involve administering antibodies of the invention to an animal,preferably a mammal, and most preferably a human, patient for treatingone or more of the disclosed diseases, disorders, or conditions.Therapeutic compounds of the invention include, but are not limited to,antibodies of the invention (including fragments, analogs andderivatives thereof as described herein) and nucleic acids encodingantibodies of the invention (including fragments, analogs andderivatives thereof and anti-idiotypic antibodies as described herein).The antibodies of the invention can be used to treat, inhibit or preventdiseases, disorders or conditions associated with aberrant expressionand/or activity of a polypeptide of the invention, including, but notlimited to, any one or more of the diseases, disorders, or conditionsdescribed herein. The treatment and/or prevention of diseases,disorders, or conditions associated with aberrant expression and/oractivity of a polypeptide of the invention includes, but is not limitedto, alleviating symptoms associated with those diseases, disorders orconditions. Antibodies of the invention may be provided inpharmaceutically acceptable compositions as known in the art or asdescribed herein.

A summary of the ways in which the antibodies of the present inventionmay be used therapeutically includes binding polynucleotides orpolypeptides of the present invention locally or systemically in thebody or by direct cytotoxicity of the antibody, e.g. as mediated bycomplement (CDC) or by effector cells (ADCC). Some of these approachesare described in more detail below. Armed with the teachings providedherein, one of ordinary skill in the art will know how to use theantibodies of the present invention for diagnostic, monitoring ortherapeutic purposes without undue experimentation.

The antibodies of this invention may be advantageously utilized incombination with other monoclonal or chimeric antibodies, or withlymphokines or hematopoietic growth factors (such as, e.g., IL-2, IL-3and IL-7), for example, which serve to increase the number or activityof effector cells which interact with the antibodies.

The antibodies of the invention may be administered alone or incombination with other types of treatments (e.g., radiation therapy,chemotherapy, hormonal therapy, immunotherapy and anti-tumor agents).Generally, administration of products of a species origin or speciesreactivity (in the case of antibodies) that is the same species as thatof the patient is preferred. Thus, in a preferred embodiment, humanantibodies, fragments derivatives, analogs, or nucleic acids, areadministered to a human patient for therapy or prophylaxis.

It is preferred to use high affinity and/or potent in vivo inhibitingand/or neutralizing antibodies against polypeptides or polynucleotidesof the present invention, fragments or regions thereof, for bothimmunoassays directed to and therapy of disorders related topolynucleotides or polypeptides, including fragments thereof, of thepresent invention. Such antibodies, fragments, or regions, willpreferably have an affinity for polynucleotides or polypeptides of theinvention, including fragments thereof. Preferred binding affinitiesinclude those with a dissociation constant or Kd less than 5×10-2 M,10-2 M, 5×10-3 M, 10-3 M, 5×10-4 M, 10-4 M, 5×10-5 M, 10-5 M, 5×10-6 M,10-6 M, 5×10-7 M. 10-7 M, 5×10-8 M, 10-8 M, 5×10-9 M, 10-9 M, 5×10-10 M,10-10 M, 5×10-11 M, 10-11 M, 5×10-12 M, 10-12 M, 5×10-13 M, 10-13 M,5×10-14 M, 10-14 M, 5×10-15 M, and 10-15 M.

Antibodies directed against polypeptides of the present invention areuseful for inhibiting allergic reactions in animals. For example, byadministering a therapeutically acceptable dose of an antibody, orantibodies, of the present invention, or a cocktail of the presentantibodies, or in combination with other antibodies of varying sources,the animal may not elicit an allergic response to antigens.

Likewise, one could envision cloning the gene encoding an antibodydirected against a polypeptide of the present invention, saidpolypeptide having the potential to elicit an allergic and/or immuneresponse in an organism, and transforming the organism with saidantibody gene such that it is expressed (e.g., constitutively,inducibly, etc.) in the organism. Thus, the organism would effectivelybecome resistant to an allergic response resulting from the ingestion orpresence of such an immune/allergic reactive polypeptide. Moreover, sucha use of the antibodies of the present invention may have particularutility in preventing and/or ameliorating autoimmune diseases and/ordisorders, as such conditions are typically a result of antibodies beingdirected against endogenous proteins. For example, in the instance wherethe polypeptide of the present invention is responsible for modulatingthe immune response to auto-antigens, transforming the organism and/orindividual with a construct comprising any of the promoters disclosedherein or otherwise known in the art, in addition, to a polynucleotideencoding the antibody directed against the polypeptide of the presentinvention could effective inhibit the organisms immune system fromeliciting an immune response to the auto-antigen(s). Detaileddescriptions of therapeutic and/or gene therapy applications of thepresent invention are provided elsewhere herein.

Alternatively, antibodies of the present invention could be produced ina plant (e.g., cloning the gene of the antibody directed against apolypeptide of the present invention, and transforming a plant with asuitable vector comprising said gene for constitutive expression of theantibody within the plant), and the plant subsequently ingested by ananimal, thereby conferring temporary immunity to the animal for thespecific antigen the antibody is directed towards (See, for example,U.S. Pat. Nos. 5,914,123 and 6,034,298).

In another embodiment, antibodies of the present invention, preferablypolyclonal antibodies, more preferably monoclonal antibodies, and mostpreferably single-chain antibodies, can be used as a means of inhibitinggene expression of a particular gene, or genes, in a human, mammal,and/or other organism. See, for example, International PublicationNumber WO 00/05391, published Feb. 3, 2000, to Dow Agrosciences LLC. Theapplication of such methods for the antibodies of the present inventionare known in the art, and are more particularly described elsewhereherein.

In yet another embodiment, antibodies of the present invention may beuseful for multimerizing the polypeptides of the present invention. Forexample, certain proteins may confer enhanced biological activity whenpresent in a multimeric state (i.e., such enhanced activity may be dueto the increased effective concentration of such proteins whereby moreprotein is available in a localized location).

Antibody-Based Gene Therapy

In a specific embodiment, nucleic acids comprising sequences encodingantibodies or functional derivatives thereof, are administered to treat,inhibit or prevent a disease or disorder associated with aberrantexpression and/or activity of a polypeptide of the invention, by way ofgene therapy. Gene therapy refers to therapy performed by theadministration to a subject of an expressed or expressible nucleic acid.In this embodiment of the invention, the nucleic acids produce theirencoded protein that mediates a therapeutic effect.

Any of the methods for gene therapy available in the art can be usedaccording to the present invention. Exemplary methods are describedbelow.

For general reviews of the methods of gene therapy, see Goldspiel etal., Clinical Pharmacy 12:488-505 (1993); Wu and Wu, Biotherapy 3:87-95(1991); Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32:573-596 (1993);Mulligan, Science 260:926-932 (1993); and Morgan and Anderson, Ann. Rev.Biochem. 62:191-217 (1993); May, TIBTECH 11(5):155-215 (1993). Methodscommonly known in the art of recombinant DNA technology which can beused are described in Ausubel et al. (eds.), Current Protocols inMolecular Biology, John Wiley & Sons, NY (1993); and Kriegler, GeneTransfer and Expression, A Laboratory Manual, Stockton Press, NY (1990).

In a preferred aspect, the compound comprises nucleic acid sequencesencoding an antibody, said nucleic acid sequences being part ofexpression vectors that express the antibody or fragments or chimericproteins or heavy or light chains thereof in a suitable host. Inparticular, such nucleic acid sequences have promoters operably linkedto the antibody coding region, said promoter being inducible orconstitutive, and, optionally, tissue- specific. In another particularembodiment, nucleic acid molecules are used in which the antibody codingsequences and any other desired sequences are flanked by regions thatpromote homologous recombination at a desired site in the genome, thusproviding for intrachromosomal expression of the antibody encodingnucleic acids (Koller and Smithies, Proc. Natl. Acad. Sci. USA86:8932-8935 (1989); Zijlstra et al., Nature 342:435-438 (1989). Inspecific embodiments, the expressed antibody molecule is a single chainantibody; alternatively, the nucleic acid sequences include sequencesencoding both the heavy and light chains, or fragments thereof, of theantibody.

Delivery of the nucleic acids into a patient may be either direct, inwhich case the patient is directly exposed to the nucleic acid ornucleic acid- carrying vectors, or indirect, in which case, cells arefirst transformed with the nucleic acids in vitro, then transplantedinto the patient. These two approaches are known, respectively, as invivo or ex vivo gene therapy.

In a specific embodiment, the nucleic acid sequences are directlyadministered in vivo, where it is expressed to produce the encodedproduct. This can be accomplished by any of numerous methods known inthe art, e.g., by constructing them as part of an appropriate nucleicacid expression vector and administering it so that they becomeintracellular, e.g., by infection using defective or attenuatedretrovirals or other viral vectors (see U.S. Pat. No. 4,980,286), or bydirect injection of naked DNA, or by use of microparticle bombardment(e.g., a gene gun; Biolistic, Dupont), or coating with lipids orcell-surface receptors or transfecting agents, encapsulation inliposomes, microparticles, or microcapsules, or by administering them inlinkage to a peptide which is known to enter the nucleus, byadministering it in linkage to a ligand subject to receptor-mediatedendocytosis (see, e.g., Wu and Wu, J. Biol. Chem. 262:4429-4432 (1987))(which can be used to target cell types specifically expressing thereceptors), etc. In another embodiment, nucleic acid-ligand complexescan be formed in which the ligand comprises a fusogenic viral peptide todisrupt endosomes, allowing the nucleic acid to avoid lysosomaldegradation. In yet another embodiment, the nucleic acid can be targetedin vivo for cell specific uptake and expression, by targeting a specificreceptor (see, e.g., PCT Publications WO 92/06180; WO 92/22635;WO92/20316; WO93/14188, WO 93/20221). Alternatively, the nucleic acidcan be introduced intracellularly and incorporated within host cell DNAfor expression, by homologous recombination (Koller and Smithies, Proc.Natl. Acad. Sci. USA 86:8932-8935 (1989); Zijlstra et al., Nature342:435-438 (1989)).

In a specific embodiment, viral vectors that contains nucleic acidsequences encoding an antibody of the invention are used. For example, aretroviral vector can be used (see Miller et al., Meth. Enzymol.217:581-599 (1993)). These retroviral vectors contain the componentsnecessary for the correct packaging of the viral genome and integrationinto the host cell DNA. The nucleic acid sequences encoding the antibodyto be used in gene therapy are cloned into one or more vectors, whichfacilitates delivery of the gene into a patient. More detail aboutretroviral vectors can be found in Boesen et al., Biotherapy 6:291-302(1994), which describes the use of a retroviral vector to deliver themdr1 gene to hematopoietic stem cells in order to make the stem cellsmore resistant to chemotherapy. Other references illustrating the use ofretroviral vectors in gene therapy are: Clowes et al., J. Clin. Invest.93:644-651 (1994); Kiem et al., Blood 83:1467-1473 (1994); Salmons andGunzberg, Human Gene Therapy 4:129-141 (1993); and Grossman and Wilson,Curr. Opin. in Genetics and Devel. 3:110-114 (1993).

Adenoviruses are other viral vectors that can be used in gene therapy.Adenoviruses are especially attractive vehicles for delivering genes torespiratory epithelia. Adenoviruses naturally infect respiratoryepithelia where they cause a mild disease. Other targets foradenovirus-based delivery systems are liver, the central nervous system,endothelial cells, and muscle. Adenoviruses have the advantage of beingcapable of infecting non-dividing cells. Kozarsky and Wilson, CurrentOpinion in Genetics and Development 3:499-503 (1993) present a review ofadenovirus-based gene therapy. Bout et al., Human Gene Therapy 5:3-10(1994) demonstrated the use of adenovirus vectors to transfer genes tothe respiratory epithelia of rhesus monkeys. Other instances of the useof adenoviruses in gene therapy can be found in Rosenfeld et al.,Science 252:431-434 (1991); Rosenfeld et al., Cell 68:143- 155 (1992);Mastrangeli et al., J. Clin. Invest. 91:225-234 (1993); PCT PublicationWO94/12649; and Wang, et al., Gene Therapy 2:775-783 (1995). In apreferred embodiment, adenovirus vectors are used.

Adeno-associated virus (AAV) has also been proposed for use in genetherapy (Walsh et al., Proc. Soc. Exp. Biol. Med. 204:289-300 (1993);U.S. Pat. No. 5,436,146).

Another approach to gene therapy involves transferring a gene to cellsin tissue culture by such methods as electroporation, lipofection,calcium phosphate mediated transfection, or viral infection. Usually,the method of transfer includes the transfer of a selectable marker tothe cells. The cells are then placed under selection to isolate thosecells that have taken up and are expressing the transferred gene. Thosecells are then delivered to a patient.

In this embodiment, the nucleic acid is introduced into a cell prior toadministration in vivo of the resulting recombinant cell. Suchintroduction can be carried out by any method known in the art,including but not limited to transfection, electroporation,microinjection, infection with a viral or bacteriophage vectorcontaining the nucleic acid sequences, cell fusion, chromosome-mediatedgene transfer, microcell-mediated gene transfer, spheroplast fusion,etc. Numerous techniques are known in the art for the introduction offoreign genes into cells (see, e.g., Loeffler and Behr, Meth. Enzymol.217:599-618 (1993); Cohen et al., Meth. Enzymol. 217:618-644 (1993);Cline, Pharmac. Ther. 29:69-92m (1985) and may be used in accordancewith the present invention, provided that the necessary developmentaland physiological functions of the recipient cells are not disrupted.The technique should provide for the stable transfer of the nucleic acidto the cell, so that the nucleic acid is expressible by the cell andpreferably heritable and expressible by its cell progeny.

The resulting recombinant cells can be delivered to a patient by variousmethods known in the art. Recombinant blood cells (e.g., hematopoieticstem or progenitor cells) are preferably administered intravenously. Theamount of cells envisioned for use depends on the desired effect,patient state, etc., and can be determined by one skilled in the art.

Cells into which a nucleic acid can be introduced for purposes of genetherapy encompass any desired, available cell type, and include but arenot limited to epithelial cells, endothelial cells, keratinocytes,fibroblasts, muscle cells, hepatocytes; blood cells such asTlymphocytes, Blymphocytes, monocytes, macrophages, neutrophils,eosinophils, megakaryocytes, granulocytes; various stem or progenitorcells, in particular hematopoietic stem or progenitor cells, e.g., asobtained from bone marrow, umbilical cord blood, peripheral blood, fetalliver, etc.

In a preferred embodiment, the cell used for gene therapy is autologousto the patient.

In an embodiment in which recombinant cells are used in gene therapy,nucleic acid sequences encoding an antibody are introduced into thecells such that they are expressible by the cells or their progeny, andthe recombinant cells are then administered in vivo for therapeuticeffect. In a specific embodiment, stem or progenitor cells are used. Anystem and/or progenitor cells which can be isolated and maintained invitro can potentially be used in accordance with this embodiment of thepresent invention (see e.g. PCT Publication WO 94/08598; Stemple andAnderson, Cell 71:973-985 (1992); Rheinwald, Meth. Cell Bio. 21A:229(1980); and Pittelkow and Scott, Mayo Clinic Proc. 61:771 (1986)).

In a specific embodiment, the nucleic acid to be introduced for purposesof gene therapy comprises an inducible promoter operably linked to thecoding region, such that expression of the nucleic acid is controllableby controlling the presence or absence of the appropriate inducer oftranscription. Demonstration of Therapeutic or Prophylactic Activity

The compounds or pharmaceutical compositions of the invention arepreferably tested in vitro, and then in vivo for the desired therapeuticor prophylactic activity, prior to use in humans. For example, in vitroassays to demonstrate the therapeutic or prophylactic utility of acompound or pharmaceutical composition include, the effect of a compoundon a cell line or a patient tissue sample. The effect of the compound orcomposition on the cell line and/or tissue sample can be determinedutilizing techniques known to those of skill in the art including, butnot limited to, rosette formation assays and cell lysis assays. Inaccordance with the invention, in vitro assays which can be used todetermine whether administration of a specific compound is indicated,include in vitro cell culture assays in which a patient tissue sample isgrown in culture, and exposed to or otherwise administered a compound,and the effect of such compound upon the tissue sample is observed.

Therapeutic/Prophylactic Administration and Compositions

The invention provides methods of treatment, inhibition and prophylaxisby administration to a subject of an effective amount of a compound orpharmaceutical composition of the invention, preferably an antibody ofthe invention. In a preferred aspect, the compound is substantiallypurified (e.g., substantially free from substances that limit its effector produce undesired side-effects). The subject is preferably an animal,including but not limited to animals such as cows, pigs, horses,chickens, cats, dogs, etc., and is preferably a mammal, and mostpreferably human.

Formulations and methods of administration that can be employed when thecompound comprises a nucleic acid or an immunoglobulin are describedabove; additional appropriate formulations and routes of administrationcan be selected from among those described herein below.

Various delivery systems are known and can be used to administer acompound of the invention, e.g., encapsulation in liposomes,microparticles, microcapsules, recombinant cells capable of expressingthe compound, receptor-mediated endocytosis (see, e.g., Wu and Wu, J.Biol. Chem. 262:4429-4432 (1987)), construction of a nucleic acid aspart of a retroviral or other vector, etc. Methods of introductioninclude but are not limited to intradermal, intramuscular,intraperitoneal, intravenous, subcutaneous, intranasal, epidural, andoral routes. The compounds or compositions may be administered by anyconvenient route, for example by infusion or bolus injection, byabsorption through epithelial or mucocutaneous linings (e.g., oralmucosa, rectal and intestinal mucosa, etc.) and may be administeredtogether with other biologically active agents. Administration can besystemic or local. In addition, it may be desirable to introduce thepharmaceutical compounds or compositions of the invention into thecentral nervous system by any suitable route, including intraventricularand intrathecal injection; intraventricular injection may be facilitatedby an intraventricular catheter, for example, attached to a reservoir,such as an Ommaya reservoir. Pulmonary administration can also beemployed, e.g., by use of an inhaler or nebulizer, and formulation withan aerosolizing agent.

In a specific embodiment, it may be desirable to administer thepharmaceutical compounds or compositions of the invention locally to thearea in need of treatment; this may be achieved by, for example, and notby way of limitation, local infusion during surgery, topicalapplication, e.g., in conjunction with a wound dressing after surgery,by injection, by means of a catheter, by means of a suppository, or bymeans of an implant, said implant being of a porous, non-porous, orgelatinous material, including membranes, such as sialastic membranes,or fibers. Preferably, when administering a protein, including anantibody, of the invention, care must be taken to use materials to whichthe protein does not absorb.

In another embodiment, the compound or composition can be delivered in avesicle, in particular a liposome (see Langer, Science 249:1527-1533(1990); Treat et al., in Liposomes in the Therapy of Infectious Diseaseand Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989); Lopez-Berestein, ibid., pp. 317-327; see generally ibid.)

In yet another embodiment, the compound or composition can be deliveredin a controlled release system. In one embodiment, a pump may be used(see Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987);Buchwald et al., Surgery 88:507 (1980); Saudek et al., N. Engl. J. Med.321:574 (1989)). In another embodiment, polymeric materials can be used(see Medical Applications of Controlled Release, Langer and Wise (eds.),CRC Pres., Boca Raton, Fla. (1974); Controlled Drug Bioavailability,Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, N.Y.(1984); Ranger and Peppas, J., Macromol. Sci. Rev. Macromol. Chem. 23:61(1983); see also Levy et al., Science 228:190 (1985); During et al.,Ann. Neurol. 25:351 (1989); Howard et al., J. Neurosurg. 71:105 (1989)).In yet another embodiment, a controlled release system can be placed inproximity of the therapeutic target, i.e., the brain, thus requiringonly a fraction of the systemic dose (see, e.g., Goodson, in MedicalApplications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)).

Other controlled release systems are discussed in the review by Langer(Science 249:1527-1533 (1990)).

In a specific embodiment where the compound of the invention is anucleic acid encoding a protein, the nucleic acid can be administered invivo to promote expression of its encoded protein, by constructing it aspart of an appropriate nucleic acid expression vector and administeringit so that it becomes intracellular, e.g., by use of a retroviral vector(see U.S. Pat. No. 4,980,286), or by direct injection, or by use ofmicroparticle bombardment (e.g., a gene gun; Biolistic, Dupont), orcoating with lipids or cell-surface receptors or transfecting agents, orby administering it in linkage to a homeobox-like peptide which is knownto enter the nucleus (see e.g., Joliot et al., Proc. Natl. Acad. Sci.USA 88:1864-1868 (1991)), etc. Alternatively, a nucleic acid can beintroduced intracellularly and incorporated within host cell DNA forexpression, by homologous recombination.

The present invention also provides pharmaceutical compositions. Suchcompositions comprise a therapeutically effective amount of a compound,and a pharmaceutically acceptable carrier. In a specific embodiment, theterm “pharmaceutically acceptable” means approved by a regulatory agencyof the Federal or a state government or listed in the U.S. Pharmacopeiaor other generally recognized pharmacopeia for use in animals, and moreparticularly in humans. The term “carrier” refers to a diluent,adjuvant, excipient, or vehicle with which the therapeutic isadministered. Such pharmaceutical carriers can be sterile liquids, suchas water and oils, including those of petroleum, animal, vegetable orsynthetic origin, such as peanut oil, soybean oil, mineral oil, sesameoil and the like. Water is a preferred carrier when the pharmaceuticalcomposition is administered intravenously. Saline solutions and aqueousdextrose and glycerol solutions can also be employed as liquid carriers,particularly for injectable solutions. Suitable pharmaceuticalexcipients include starch, glucose, lactose, sucrose, gelatin, malt,rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate,talc, sodium chloride, dried skim milk, glycerol, propylene, glycol,water, ethanol and the like. The composition, if desired, can alsocontain minor amounts of wetting or emulsifying agents, or pH bufferingagents. These compositions can take the form of solutions, suspensions,emulsion, tablets, pills, capsules, powders, sustained-releaseformulations and the like. The composition can be formulated as asuppository, with traditional binders and carriers such astriglycerides. Oral formulation can include standard carriers such aspharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharine, cellulose, magnesium carbonate, etc. Examples ofsuitable pharmaceutical carriers are described in “Remington'sPharmaceutical Sciences” by E. W. Martin. Such compositions will containa therapeutically effective amount of the compound, preferably inpurified form, together with a suitable amount of carrier so as toprovide the form for proper administration to the patient. Theformulation should suit the mode of administration.

In a preferred embodiment, the composition is formulated in accordancewith routine procedures as a pharmaceutical composition adapted forintravenous administration to human beings. Typically, compositions forintravenous administration are solutions in sterile isotonic aqueousbuffer. Where necessary, the composition may also include a solubilizingagent and a local anesthetic such as lignocaine to ease pain at the siteof the injection. Generally, the ingredients are supplied eitherseparately or mixed together in unit dosage form, for example, as a drylyophilized powder or water free concentrate in a hermetically sealedcontainer such as an ampoule or sachette indicating the quantity ofactive agent. Where the composition is to be administered by infusion,it can be dispensed with an infusion bottle containing sterilepharmaceutical grade water or saline. Where the composition isadministered by injection, an ampoule of sterile water for injection orsaline can be provided so that the ingredients may be mixed prior toadministration.

The compounds of the invention can be formulated as neutral or saltforms. Pharmaceutically acceptable salts include those formed withanions such as those derived from hydrochloric, phosphoric, acetic,oxalic, tartaric acids, etc., and those formed with cations such asthose derived from sodium, potassium, ammonium, calcium, ferrichydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol,histidine, procaine, etc.

The amount of the compound of the invention which will be effective inthe treatment, inhibition and prevention of a disease or disorderassociated with aberrant expression and/or activity of a polypeptide ofthe invention can be determined by standard clinical techniques. Inaddition, in vitro assays may optionally be employed to help identifyoptimal dosage ranges. The precise dose to be employed in theformulation will also depend on the route of administration, and theseriousness of the disease or disorder, and should be decided accordingto the judgment of the practitioner and each patient's circumstances.Effective doses may be extrapolated from dose-response curves derivedfrom in vitro or animal model test systems.

For antibodies, the dosage administered to a patient is typically 0.1mg/kg to 100 mg/kg of the patient's body weight. Preferably, the dosageadministered to a patient is between 0.1 mg/kg and 20 mg/kg of thepatient's body weight, more preferably 1 mg/kg to 10 mg/kg of thepatient's body weight. Generally, human antibodies have a longerhalf-life within the human body than antibodies from other species dueto the immune response to the foreign polypeptides. Thus, lower dosagesof human antibodies and less frequent administration is often possible.Further, the dosage and frequency of administration of antibodies of theinvention may be reduced by enhancing uptake and tissue penetration(e.g., into the brain) of the antibodies by modifications such as, forexample, lipidation.

The invention also provides a pharmaceutical pack or kit comprising oneor more containers filled with one or more of the ingredients of thepharmaceutical compositions of the invention. Optionally associated withsuch container(s) can be a notice in the form prescribed by agovernmental agency regulating the manufacture, use or sale ofpharmaceuticals or biological products, which notice reflects approvalby the agency of manufacture, use or sale for human administration.

Diagnosis and Imaging With Antibodies

Labeled antibodies, and derivatives and analogs thereof, whichspecifically bind to a polypeptide of interest can be used fordiagnostic purposes to detect, diagnose, or monitor diseases, disorders,and/or conditions associated with the aberrant expression and/oractivity of a polypeptide of the invention. The invention provides forthe detection of aberrant expression of a polypeptide of interest,comprising (a) assaying the expression of the polypeptide of interest incells or body fluid of an individual using one or more antibodiesspecific to the polypeptide interest and (b) comparing the level of geneexpression with a standard gene expression level, whereby an increase ordecrease in the assayed polypeptide gene expression level compared tothe standard expression level is indicative of aberrant expression.

The invention provides a diagnostic assay for diagnosing a disorder,comprising (a) assaying the expression of the polypeptide of interest incells or body fluid of an individual using one or more antibodiesspecific to the polypeptide interest and (b) comparing the level of geneexpression with a standard gene expression level, whereby an increase ordecrease in the assayed polypeptide gene expression level compared tothe standard expression level is indicative of a particular disorder.With respect to cancer, the presence of a relatively high amount oftranscript in biopsied tissue from an individual may indicate apredisposition for the development of the disease, or may provide ameans for detecting the disease prior to the appearance of actualclinical symptoms. A more definitive diagnosis of this type may allowhealth professionals to employ preventative measures or aggressivetreatment earlier thereby preventing the development or furtherprogression of the cancer.

Antibodies of the invention can be used to assay protein levels in abiological sample using classical immunohistological methods known tothose of skill in the art (e.g., see Jalkanen, et al., J. Cell. Biol.101:976-985 (1985); Jalkanen, et al., J. Cell. Biol. 105:3087-3096(1987)). Other antibody-based methods useful for detecting protein geneexpression include immunoassays, such as the enzyme linked immunosorbentassay (ELISA) and the radioimmunoassay (RIA). Suitable antibody assaylabels are known in the art and include enzyme labels, such as, glucoseoxidase; radioisotopes, such as iodine (125I, 121I), carbon (14C),sulfur (35S), tritium (3H), indium (112In), and technetium (99Tc);luminescent labels, such as luminol; and fluorescent labels, such asfluorescein and rhodamine, and biotin.

One aspect of the invention is the detection and diagnosis of a diseaseor disorder associated with aberrant expression of a polypeptide ofinterest in an animal, preferably a mammal and most preferably a human.In one embodiment, diagnosis comprises: a) administering (for example,parenterally, subcutaneously, or intraperitoneally) to a subject aneffective amount of a labeled molecule which specifically binds to thepolypeptide of interest; b) waiting for a time interval following theadministering for permitting the labeled molecule to preferentiallyconcentrate at sites in the subject where the polypeptide is expressed(and for unbound labeled molecule to be cleared to background level); c)determining background level; and d) detecting the labeled molecule inthe subject, such that detection of labeled molecule above thebackground level indicates that the subject has a particular disease ordisorder associated with aberrant expression of the polypeptide ofinterest. Background level can be determined by various methodsincluding, comparing the amount of labeled molecule detected to astandard value previously determined for a particular system.

It will be understood in the art that the size of the subject and theimaging system used will determine the quantity of imaging moiety neededto produce diagnostic images. In the case of a radioisotope moiety, fora human subject, the quantity of radioactivity injected will normallyrange from about 5 to 20 millicuries of 99mTc. The labeled antibody orantibody fragment will then preferentially accumulate at the location ofcells which contain the specific protein. In vivo tumor imaging isdescribed in S. W. Burchiel et al., “Immunopharmacokinetics ofRadiolabeled Antibodies and Their Fragments.” (Chapter 13 in TumorImaging: The Radiochemical Detection of Cancer, S. W. Burchiel and B. A.Rhodes, eds., Masson Publishing Inc. (1982).

Depending on several variables, including the type of label used and themode of administration, the time interval following the administrationfor permitting the labeled molecule to preferentially concentrate atsites in the subject and for unbound labeled molecule to be cleared tobackground level is 6 to 48 hours or 6 to 24 hours or 6 to 12 hours. Inanother embodiment the time interval following administration is 5 to 20days or 5 to 10 days.

In an embodiment, monitoring of the disease or disorder is carried outby repeating the method for diagnosing the disease or disease, forexample, one month after initial diagnosis, six months after initialdiagnosis, one year after initial diagnosis, etc.

Presence of the labeled molecule can be detected in the patient usingmethods known in the art for in vivo scanning. These methods depend uponthe type of label used. Skilled artisans will be able to determine theappropriate method for detecting a particular label. Methods and devicesthat may be used in the diagnostic methods of the invention include, butare not limited to, computed tomography (CT), whole body scan such asposition emission tomography (PET), magnetic resonance imaging (MRI),and sonography.

In a specific embodiment, the molecule is labeled with a radioisotopeand is detected in the patient using a radiation responsive surgicalinstrument (Thurston et al., U.S. Pat. No. 5,441,050). In anotherembodiment, the molecule is labeled with a fluorescent compound and isdetected in the patient using a fluorescence responsive scanninginstrument. In another embodiment, the molecule is labeled with apositron emitting metal and is detected in the patent using positronemission-tomography. In yet another embodiment, the molecule is labeledwith a paramagnetic label and is detected in a patient using magneticresonance imaging (MRI).

Kits

The present invention provides kits that can be used in the abovemethods. In one embodiment, a kit comprises an antibody of theinvention, preferably a purified antibody, in one or more containers. Ina specific embodiment, the kits of the present invention contain asubstantially isolated polypeptide comprising an epitope which isspecifically immunoreactive with an antibody included in the kit.Preferably, the kits of the present invention further comprise a controlantibody which does not react with the polypeptide of interest. Inanother specific embodiment, the kits of the present invention contain ameans for detecting the binding of an antibody to a polypeptide ofinterest (e.g., the antibody may be conjugated to a detectable substratesuch as a fluorescent compound, an enzymatic substrate, a radioactivecompound or a luminescent compound, or a second antibody whichrecognizes the first antibody may be conjugated to a detectablesubstrate).

In another specific embodiment of the present invention, the kit is adiagnostic kit for use in screening serum containing antibodies specificagainst proliferative and/or cancerous polynucleotides and polypeptides.Such a kit may include a control antibody that does not react with thepolypeptide of interest. Such a kit may include a substantially isolatedpolypeptide antigen comprising an epitope which is specificallyimmunoreactive with at least one anti-polypeptide antigen antibody.Further, such a kit includes means for detecting the binding of saidantibody to the antigen (e.g., the antibody may be conjugated to afluorescent compound such as fluorescein or rhodamine which can bedetected by flow cytometry). In specific embodiments, the kit mayinclude a recombinantly produced or chemically synthesized polypeptideantigen. The polypeptide antigen of the kit may also be attached to asolid support.

In a more specific embodiment the detecting means of the above-describedkit includes a solid support to which said polypeptide antigen isattached. Such a kit may also include a non-attached reporter-labeledanti-human antibody. In this embodiment, binding of the antibody to thepolypeptide antigen can be detected by binding of the saidreporter-labeled antibody.

In an additional embodiment, the invention includes a diagnostic kit foruse in screening serum containing antigens of the polypeptide of theinvention. The diagnostic kit includes a substantially isolated antibodyspecifically immunoreactive with polypeptide or polynucleotide antigens,and means for detecting the binding of the polynucleotide or polypeptideantigen to the antibody. In one embodiment, the antibody is attached toa solid support. In a specific embodiment, the antibody may be amonoclonal antibody. The detecting means of the kit may include asecond, labeled monoclonal antibody. Alternatively, or in addition, thedetecting means may include a labeled, competing antigen.

In one diagnostic configuration, test serum is reacted with a solidphase reagent having a surface-bound antigen obtained by the methods ofthe present invention. After binding with specific antigen antibody tothe reagent and removing unbound serum components by washing, thereagent is reacted with reporter-labeled anti-human antibody to bindreporter to the reagent in proportion to the amount of boundanti-antigen antibody on the solid support. The reagent is again washedto remove unbound labeled antibody, and the amount of reporterassociated with the reagent is determined. Typically, the reporter is anenzyme which is detected by incubating the solid phase in the presenceof a suitable fluorometric, luminescent or colorimetric substrate(Sigma, St. Louis, Mo.).

The solid surface reagent in the above assay is prepared by knowntechniques for attaching protein material to solid support material,such as polymeric beads, dip sticks, 96-well plate or filter material.These attachment methods generally include non-specific adsorption ofthe protein to the support or covalent attachment of the protein,typically through a free amine group, to a chemically reactive group onthe solid support, such as an activated carboxyl, hydroxyl, or aldehydegroup. Alternatively, streptavidin coated plates can be used inconjunction with biotinylated antigen(s).

Thus, the invention provides an assay system or kit for carrying outthis diagnostic method. The kit generally includes a support withsurface- bound recombinant antigens, and a reporter-labeled anti-humanantibody for detecting surface-bound anti-antigen antibody.

Fusion Proteins

Any polypeptide of the present invention can be used to generate fusionproteins. For example, the polypeptide of the present invention, whenfused to a second protein, can be used as an antigenic tag. Antibodiesraised against the polypeptide of the present invention can be used toindirectly detect the second protein by binding to the polypeptide.Moreover, because certain proteins target cellular locations based ontrafficking signals, the polypeptides of the present invention can beused as targeting molecules once fused to other proteins.

Examples of domains that can be fused to polypeptides of the presentinvention include not only heterologous signal sequences, but also otherheterologous functional regions. The fusion does not necessarily need tobe direct, but may occur through linker sequences.

Moreover, fusion proteins may also be engineered to improvecharacteristics of the polypeptide of the present invention. Forinstance, a region of additional amino acids, particularly charged aminoacids, may be added to the N-terminus of the polypeptide to improvestability and persistence during purification from the host cell orsubsequent handling and storage. Peptide moieties may be added to thepolypeptide to facilitate purification. Such regions may be removedprior to final preparation of the polypeptide. Similarly, peptidecleavage sites can be introduced in-between such peptide moieties, whichcould additionally be subjected to protease activity to remove saidpeptide(s) from the protein of the present invention. The addition ofpeptide moieties, including peptide cleavage sites, to facilitatehandling of polypeptides are familiar and routine techniques in the art.

Moreover, polypeptides of the present invention, including fragments,and specifically epitopes, can be combined with parts of the constantdomain of immunoglobulins (IgA, IgE, IgG, IgM) or portions thereof (CH1,CH2, CH3, and any combination thereof, including both entire domains andportions thereof), resulting in chimeric polypeptides. These fusionproteins facilitate purification and show an increased half-life invivo. One reported example describes chimeric proteins consisting of thefirst two domains of the human CD4-polypeptide and various domains ofthe constant regions of the heavy or light chains of mammalianimmunoglobulins. (EP A 394,827; Traunecker et al., Nature 331:84-86(1988).) Fusion proteins having disulfide-linked dimeric structures (dueto the IgG) can also be more efficient in binding and neutralizing othermolecules, than the monomeric secreted protein or protein fragmentalone. (Fountoulakis et al., J. Biochem. 270:3958-3964 (1995).)

Similarly, EP-A-O 464 533 (Canadian counterpart 2045869) disclosesfusion proteins comprising various portions of the constant region ofimmunoglobulin molecules together with another human protein or partthereof. In many cases, the Fc part in a fusion protein is beneficial intherapy and diagnosis, and thus can result in, for example, improvedpharmacokinetic properties. (EP-A 0232 262.) Alternatively, deleting theFc part after the fusion protein has been expressed, detected, andpurified, would be desired. For example, the Fc portion may hindertherapy and diagnosis if the fusion protein is used as an antigen forimmunizations. In drug discovery, for example, human proteins, such ashIL-5, have been fused with Fc portions for the purpose ofhigh-throughput screening assays to identify antagonists of hIL-5. (See,D. Bennett et al., J. Molecular Recognition 8:52-58 (1995); K. Johansonet al., J. Biol. Chem. 270:9459-9471 (1995).)

Moreover, the polypeptides of the present invention can be fused tomarker sequences (also referred to as “tags”). Due to the availabilityof antibodies specific to such “tags”, purification of the fusedpolypeptide of the invention, and/or its identification is significantlyfacilitated since antibodies specific to the polypeptides of theinvention are not required. Such purification may be in the form of anaffinity purification whereby an anti-tag antibody or another type ofaffinity matrix (e.g., anti-tag antibody attached to the matrix of aflow-thru column) that binds to the epitope tag is present. In preferredembodiments, the marker amino acid sequence is a hexa-histidine peptide,such as the tag provided in a pQE vector (QIAGEN, Inc., 9259 EtonAvenue, Chatsworth, Calif., 91311), among others, many of which arecommercially available. As described in Gentz et al., Proc. Natl. Acad.Sci. USA 86:821-824 (1989), for instance, hexa-histidine provides forconvenient purification of the fusion protein. Another peptide taguseful for purification, the “HA” tag, corresponds to an epitope derivedfrom the influenza hemagglutinin protein. (Wilson et al., Cell 37:767(1984)).

The skilled artisan would acknowledge the existence of other “tags”which could be readily substituted for the tags referred to supra forpurification and/or identification of polypeptides of the presentinvention (Jones C., et al., J Chromatogr A. 707(1):3-22 (1995)). Forexample, the c-myc tag and the 8F9, 3C7, 6E10, G4m B7 and 9E10antibodies thereto (Evan et al., Molecular and Cellular Biology5:3610-3616 (1985)); the Herpes Simplex virus glycoprotein D (gD) tagand its antibody (Paborsky et al., Protein Engineering, 3(6):547-553(1990), the Flag-peptide—i.e., the octapeptide sequence DYKDDDDK (SEQ IDNO:51), (Hopp et al., Biotech. 6:1204-1210 (1988); the KT3 epitopepeptide (Martin et al., Science, 255:192-194 (1992)); a-tubulin epitopepeptide (Skinner et al., J. Biol. Chem., 266:15136-15166, (1991)); theT7 gene 10 protein peptide tag (Lutz-Freyermuth et al., Proc. Natl. Sci.USA, 87:6363-6397 (1990)), the FITC epitope (Zymed, Inc.), the GFPepitope (Zymed, Inc.), and the Rhodamine epitope (Zymed, Inc.).

The present invention also encompasses the attachment of up to ninecodons encoding a repeating series of up to nine arginine amino acids tothe coding region of a polynucleotide of the present invention. Theinvention also encompasses chemically derivitizing a polypeptide of thepresent invention with a repeating series of up to nine arginine aminoacids. Such a tag, when attached to a polypeptide, has recently beenshown to serve as a universal pass, allowing compounds access to theinterior of cells without additional derivitization or manipulation(Wender, P., et al., unpublished data).

Protein fusions involving polypeptides of the present invention,including fragments and/or variants thereof, can be used for thefollowing, non-limiting examples, subcellular localization of proteins,determination of protein-protein interactions via immunoprecipitation,purification of proteins via affinity chromatography, functional and/orstructural characterization of protein. The present invention alsoencompasses the application of hapten specific antibodies for any of theuses referenced above for epitope fusion proteins. For example, thepolypeptides of the present invention could be chemically derivatized toattach hapten molecules (e.g., DNP, (Zymed, Inc.)). Due to theavailability of monoclonal antibodies specific to such haptens, theprotein could be readily purified using immunoprecipation, for example.

Polypeptides of the present invention, including fragments and/orvariants thereof, in addition to, antibodies directed against suchpolypeptides, fragments, and/or variants, may be fused to any of anumber of known, and yet to be determined, toxins, such as ricin,saporin (Mashiba H, et al., Ann. N. Y. Acad. Sci. 1999;886:233-5), or HCtoxin (Tonukari N J, et al., Plant Cell. February 2000;12(2):237-248),for example. Such fusions could be used to deliver the toxins to desiredtissues for which a ligand or a protein capable of binding to thepolypeptides of the invention exists.

The invention encompasses the fusion of antibodies directed againstpolypeptides of the present invention, including variants and fragmentsthereof, to said toxins for delivering the toxin to specific locationsin a cell, to specific tissues, and/or to specific species. Suchbifunctional antibodies are known in the art, though a review describingadditional advantageous fusions, including citations for methods ofproduction, can be found in P. J. Hudson, Curr. Opp. In. Imm.11:548-557, (1999); this publication, in addition to the referencescited therein, are hereby incorporated by reference in their entiretyherein. In this context, the term “toxin” may be expanded to include anyheterologous protein, a small molecule, radionucleotides, cytotoxicdrugs, liposomes, adhesion molecules, glycoproteins, ligands, cell ortissue-specific ligands, enzymes, of bioactive agents, biologicalresponse modifiers, anti-fungal agents, hormones, steroids, vitamins,peptides, peptide analogs, anti-allergenic agents, anti-tubercularagents, anti-viral agents, antibiotics, anti-protozoan agents, chelates,radioactive particles, radioactive ions, X-ray contrast agents,monoclonal antibodies, polyclonal antibodies and genetic material. Inview of the present disclosure, one skilled in the art could determinewhether any particular “toxin” could be used in the compounds of thepresent invention. Examples of suitable “toxins” listed above areexemplary only and are not intended to limit the “toxins” that may beused in the present invention.

Thus, any of these above fusions can be engineered using thepolynucleotides or the polypeptides of the present invention.

Vectors, Host Cells, and Protein Production

The present invention also relates to vectors containing thepolynucleotide of the present invention, host cells, and the productionof polypeptides by recombinant techniques. The vector may be, forexample, a phage, plasmid, viral, or retroviral vector. Retroviralvectors may be replication competent or replication defective. In thelatter case, viral propagation generally will occur only incomplementing host cells.

The polynucleotides may be joined to a vector containing a selectablemarker for propagation in a host. Generally, a plasmid vector isintroduced in a precipitate, such as a calcium phosphate precipitate, orin a complex with a charged lipid. If the vector is a virus, it may bepackaged in vitro using an appropriate packaging cell line and thentransduced into host cells.

The polynucleotide insert should be operatively linked to an appropriatepromoter, such as the phage lambda PL promoter, the E. coli lac, trp,phoA and tac promoters, the SV40 early and late promoters and promotersof retroviral LTRs, to name a few. Other suitable promoters will beknown to the skilled artisan. The expression constructs will furthercontain sites for transcription initiation, termination, and, in thetranscribed region, a ribosome binding site for translation. The codingportion of the transcripts expressed by the constructs will preferablyinclude a translation initiating codon at the beginning and atermination codon (UAA, UGA or UAG) appropriately positioned at the endof the polypeptide to be translated.

As indicated, the expression vectors will preferably include at leastone selectable marker. Such markers include dihydrofolate reductase,G418 or neomycin resistance for eukaryotic cell culture andtetracycline, kanamycin or ampicillin resistance genes for culturing inE. coli and other bacteria. Representative examples of appropriate hostsinclude, but are not limited to, bacterial cells, such as E. coli,Streptomyces and Salmonella typhimurium cells; fungal cells, such asyeast cells (e.g., Saccharomyces cerevisiae or Pichia pastoris (ATCCAccession No. 201178)); insect cells such as Drosophila S2 andSpodoptera Sf9 cells; animal cells such as CHO, COS, 293, and Bowesmelanoma cells; and plant cells. Appropriate culture mediums andconditions for the above-described host cells are known in the art.

Among vectors preferred for use in bacteria include pQE70, pQE60 andpQE-9, available from QIAGEN, Inc.; pBluescript vectors, Phagescriptvectors, pNH8A, pNH16a, pNH18A, pNH46A, available from StratageneCloning Systems, Inc.; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5available from Pharmacia Biotech, Inc. Among preferred eukaryoticvectors are pWLNEO, pSV2CAT, pOG44, pXT1 and pSG available fromStratagene; and pSVK3, pBPV, pMSG and pSVL available from Pharmacia.Preferred expression vectors for use in yeast systems include, but arenot limited to pYES2, pYD1, pTEF1/Zeo, pYES2/GS, pPICZ, pGAPZ,pGAPZalph, pPIC9, pPIC3.5, pHIL-D2, pHIL-S1, pPIC3.5K, pPIC9K, andPAO815 (all available from Invitrogen, Carlsbad, Calif.). Other suitablevectors will be readily apparent to the skilled artisan.

Introduction of the construct into the host cell can be effected bycalcium phosphate transfection, DEAE-dextran mediated transfection,cationic lipid-mediated transfection, electroporation, transduction,infection, or other methods. Such methods are described in many standardlaboratory manuals, such as Davis et al., Basic Methods In MolecularBiology (1986). It is specifically contemplated that the polypeptides ofthe present invention may in fact be expressed by a host cell lacking arecombinant vector.

A polypeptide of this invention can be recovered and purified fromrecombinant cell cultures by well-known methods including ammoniumsulfate or ethanol precipitation, acid extraction, anion or cationexchange chromatography, phosphocellulose chromatography, hydrophobicinteraction chromatography, affinity chromatography, hydroxylapatitechromatography and lectin chromatography. Most preferably, highperformance liquid chromatography (“HPLC”) is employed for purification.

Polypeptides of the present invention, and preferably the secreted form,can also be recovered from: products purified from natural sources,including bodily fluids, tissues and cells, whether directly isolated orcultured; products of chemical synthetic procedures; and productsproduced by recombinant techniques from a prokaryotic or eukaryotichost, including, for example, bacterial, yeast, higher plant, insect,and mammalian cells. Depending upon the host employed in a recombinantproduction procedure, the polypeptides of the present invention may beglycosylated or may be non-glycosylated. In addition, polypeptides ofthe invention may also include an initial modified methionine residue,in some cases as a result of host-mediated processes. Thus, it is wellknown in the art that the N-terminal methionine encoded by thetranslation initiation codon generally is removed with high efficiencyfrom any protein after translation in all eukaryotic cells. While theN-terminal methionine on most proteins also is efficiently removed inmost prokaryotes, for some proteins, this prokaryotic removal process isinefficient, depending on the nature of the amino acid to which theN-terminal methionine is covalently linked.

In one embodiment, the yeast Pichia pastoris is used to express thepolypeptide of the present invention in a eukaryotic system. Pichiapastoris is a methylotrophic yeast which can metabolize methanol as itssole carbon source. A main step in the methanol metabolization pathwayis the oxidation of methanol to formaldehyde using O2. This reaction iscatalyzed by the enzyme alcohol oxidase. In order to metabolize methanolas its sole carbon source, Pichia pastoris must generate high levels ofalcohol oxidase due, in part, to the relatively low affinity of alcoholoxidase for O2. Consequently, in a growth medium depending on methanolas a main carbon source, the promoter region of one of the two alcoholoxidase genes (AOX1) is highly active. In the presence of methanol,alcohol oxidase produced from the AOX1 gene comprises up toapproximately 30% of the total soluble protein in Pichia pastoris. See,Ellis, S. B., et al., Mol. Cell. Biol. 5:1111-21 (1985); Koutz, P. J, etal., Yeast 5:167-77 (1989); Tschopp, J. F., et al., Nucl. Acids Res.15:3859-76 (1987). Thus, a heterologous coding sequence, such as, forexample, a polynucleotide of the present invention, under thetranscriptional regulation of all or part of the AOX1 regulatorysequence is expressed at exceptionally high levels in Pichia yeast grownin the presence of methanol.

In one example, the plasmid vector pPIC9K is used to express DNAencoding a polypeptide of the invention, as set forth herein, in aPichea yeast system essentially as described in “Pichia Protocols:Methods in Molecular Biology,” D. R. Higgins and J. Cregg, eds. TheHumana Press, Totowa, N.J., 1998. This expression vector allowsexpression and secretion of a protein of the invention by virtue of thestrong AOX1 promoter linked to the Pichia pastoris alkaline phosphatase(PHO) secretory signal peptide (i.e., leader) located upstream of amultiple cloning site.

Many other yeast vectors could be used in place of pPIC9K, such as,pYES2, pYD1, pTEF1/Zeo, pYES2/GS, pPICZ, pGAPZ, pGAPZalpha, pPIC9,pPIC3.5, pHIL-D2, pHIL-S1, pPIC3.5K, and PAO815, as one skilled in theart would readily appreciate, as long as the proposed expressionconstruct provides appropriately located signals for transcription,translation, secretion (if desired), and the like, including an in-frameAUG, as required.

In another embodiment, high-level expression of a heterologous codingsequence, such as, for example, a polynucleotide of the presentinvention, may be achieved by cloning the heterologous polynucleotide ofthe invention into an expression vector such as, for example, pGAPZ orpGAPZalpha, and growing the yeast culture in the absence of methanol.

In addition to encompassing host cells containing the vector constructsdiscussed herein, the invention also encompasses primary, secondary, andimmortalized host cells of vertebrate origin, particularly mammalianorigin, that have been engineered to delete or replace endogenousgenetic material (e.g., coding sequence), and/or to include geneticmaterial (e.g., heterologous polynucleotide sequences) that is operablyassociated with the polynucleotides of the invention, and whichactivates, alters, and/or amplifies endogenous polynucleotides. Forexample, techniques known in the art may be used to operably associateheterologous control regions (e.g., promoter and/or enhancer) andendogenous polynucleotide sequences via homologous recombination,resulting in the formation of a new transcription unit (see, e.g., U.S.Pat. No. 5,641,670, issued Jun. 24, 1997; U.S. Pat. No. 5,733,761,issued Mar. 31, 1998; International Publication No. WO 96/29411,published Sep. 26, 1996; International Publication No. WO 94/12650,published Aug. 4, 1994; Koller et al., Proc. Natl. Acad. Sci. USA86:8932-8935 (1989); and Zijlstra et al., Nature 342:435-438 (1989), thedisclosures of each of which are incorporated by reference in theirentireties).

In addition, polypeptides of the invention can be chemically synthesizedusing techniques known in the art (e.g., see Creighton, 1983, Proteins:Structures and Molecular Principles, W.H. Freeman & Co., N.Y., andHunkapiller et al., Nature, 310:105-111 (1984)). For example, apolypeptide corresponding to a fragment of a polypeptide sequence of theinvention can be synthesized by use of a peptide synthesizer.Furthermore, if desired, nonclassical amino acids or chemical amino acidanalogs can be introduced as a substitution or addition into thepolypeptide sequence. Non-classical amino acids include, but are notlimited to, to the D-isomers of the common amino acids,2,4-diaminobutyric acid, a-amino isobutyric acid, 4-aminobutyric acid,Abu, 2-amino butyric acid, g-Abu, e-Ahx, 6-amino hexanoic acid, Aib,2-amino isobutyric acid, 3-amino propionic acid, omithine, norleucine,norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline,cysteic acid, t-butylglycine, t-butylalanine, phenylglycine,cyclohexylalanine, b-alanine, fluoro-amino acids, designer amino acidssuch as b-methyl amino acids, Ca-methyl amino acids, Na-methyl aminoacids, and amino acid analogs in general. Furthermore, the amino acidcan be D (dextrorotary) or L (levorotary).

The invention encompasses polypeptides which are differentially modifiedduring or after translation, e.g., by glycosylation, acetylation,phosphorylation, amidation, derivatization by known protecting/blockinggroups, proteolytic cleavage, linkage to an antibody molecule or othercellular ligand, etc. Any of numerous chemical modifications may becarried out by known techniques, including but not limited, to specificchemical cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8protease, NaBH4; acetylation, formylation, oxidation, reduction;metabolic synthesis in the presence of tunicamycin; etc.

Additional post-translational modifications encompassed by the inventioninclude, for example, e.g., N-linked or O-linked carbohydrate chains,processing of N-terminal or C-terminal ends), attachment of chemicalmoieties to the amino acid backbone, chemical modifications of N-linkedor O-linked carbohydrate chains, and addition or deletion of anN-terminal methionine residue as a result of prokaryotic host cellexpression. The polypeptides may also be modified with a detectablelabel, such as an enzymatic, fluorescent, isotopic or affinity label toallow for detection and isolation of the protein, the addition ofepitope tagged peptide fragments (e.g., FLAG, HA, GST, thioredoxin,maltose binding protein, etc.), attachment of affinity tags such asbiotin and/or streptavidin, the covalent attachment of chemical moietiesto the amino acid backbone, N— or C-terminal processing of thepolypeptides ends (e.g., proteolytic processing), deletion of theN-terminal methionine residue, etc.

Also provided by the invention are chemically modified derivatives ofthe polypeptides of the invention which may provide additionaladvantages such as increased solubility, stability and circulating timeof the polypeptide, or decreased immunogenicity (see U.S. Pat. No.4,179,337). The chemical moieties for derivitization may be selectedfrom water soluble polymers such as polyethylene glycol, ethyleneglycol/propylene glycol copolymers, carboxymethylcellulose, dextran,polyvinyl alcohol and the like. The polypeptides may be modified atrandom positions within the molecule, or at predetermined positionswithin the molecule and may include one, two, three or more attachedchemical moieties.

The invention further encompasses chemical derivitization of thepolypeptides of the present invention, preferably where the chemical isa hydrophilic polymer residue. Exemplary hydrophilic polymers, includingderivatives, may be those that include polymers in which the repeatingunits contain one or more hydroxy groups (polyhydroxy polymers),including, for example, poly(vinyl alcohol); polymers in which therepeating units contain one or more amino groups (polyamine polymers),including, for example, peptides, polypeptides, proteins andlipoproteins, such as albumin and natural lipoproteins; polymers inwhich the repeating units contain one or more carboxy groups(polycarboxy polymers), including, for example, carboxymethylcellulose,alginic acid and salts thereof, such as sodium and calcium alginate,glycosaminoglycans and salts thereof, including salts of hyaluronicacid, phosphorylated and sulfonated derivatives of carbohydrates,genetic material, such as interleukin-2 and interferon, andphosphorothioate oligomers; and polymers in which the repeating unitscontain one or more saccharide moieties (polysaccharide polymers),including, for example, carbohydrates.

The molecular weight of the hydrophilic polymers may vary, and isgenerally about 50 to about 5,000,000, with polymers having a molecularweight of about 100 to about 50,000 being preferred. The polymers may bebranched or unbranched. More preferred polymers have a molecular weightof about 150 to about 10,000, with molecular weights of 200 to about8,000 being even more preferred.

For polyethylene glycol, the preferred molecular weight is between about1 kDa and about 100 kDa (the term “about” indicating that inpreparations of polyethylene glycol, some molecules will weigh more,some less, than the stated molecular weight) for ease in handling andmanufacturing. Other sizes may be used, depending on the desiredtherapeutic profile (e.g., the duration of sustained release desired,the effects, if any on biological activity, the ease in handling, thedegree or lack of antigenicity and other known effects of thepolyethylene glycol to a therapeutic protein or analog).

Additional preferred polymers which may be used to derivatizepolypeptides of the invention, include, for example, poly(ethyleneglycol) (PEG), poly(vinylpyrrolidine), polyoxomers, polysorbate andpoly(vinyl alcohol), with PEG polymers being particularly preferred.Preferred among the PEG polymers are PEG polymers having a molecularweight of from about 100 to about 10,000. More preferably, the PEGpolymers have a molecular weight of from about 200 to about 8,000, withPEG 2,000, PEG 5,000 and PEG 8,000, which have molecular weights of2,000, 5,000 and 8,000, respectively, being even more preferred. Othersuitable hydrophilic polymers, in addition to those exemplified above,will be readily apparent to one skilled in the art based on the presentdisclosure. Generally, the polymers used may include polymers that canbe attached to the polypeptides of the invention via alkylation oracylation reactions.

The polyethylene glycol molecules (or other chemical moieties) should beattached to the protein with consideration of effects on functional orantigenic domains of the protein. There are a number of attachmentmethods available to those skilled in the art, e.g., EP 0 401 384,herein incorporated by reference (coupling PEG to G-CSF), see also Maliket al., Exp. Hematol. 20:1028-1035 (1992) (reporting pegylation ofGM-CSF using tresyl chloride). For example, polyethylene glycol may becovalently bound through amino acid residues via a reactive group, suchas, a free amino or carboxyl group. Reactive groups are those to whichan activated polyethylene glycol molecule may be bound. The amino acidresidues having a free amino group may include lysine residues and theN-terminal amino acid residues; those having a free carboxyl group mayinclude aspartic acid residues glutamic acid residues and the C-terminalamino acid residue. Sulfhydryl groups may also be used as a reactivegroup for attaching the polyethylene glycol molecules. Preferred fortherapeutic purposes is attachment at an amino group, such as attachmentat the N-terminus or lysine group.

One may specifically desire proteins chemically modified at theN-terminus. Using polyethylene glycol as an illustration of the presentcomposition, one may select from a variety of polyethylene glycolmolecules (by molecular weight, branching, etc.), the proportion ofpolyethylene glycol molecules to protein (polypeptide) molecules in thereaction mix, the type of pegylation reaction to be performed, and themethod of obtaining the selected N-terminally pegylated protein. Themethod of obtaining the N-terminally pegylated preparation (i.e.,separating this moiety from other monopegylated moieties if necessary)may be by purification of the N-terminally pegylated material from apopulation of pegylated protein molecules. Selective proteins chemicallymodified at the N-terminus modification may be accomplished by reductivealkylation which exploits differential reactivity of different types ofprimary amino groups (lysine versus the N-terminus) available forderivatization in a particular protein. Under the appropriate reactionconditions, substantially selective derivatization of the protein at theN-terminus with a carbonyl group containing polymer is achieved.

As with the various polymers exemplified above, it is contemplated thatthe polymeric residues may contain functional groups in addition, forexample, to those typically involved in linking the polymeric residuesto the polypeptides of the present invention. Such functionalitiesinclude, for example, carboxyl, amine, hydroxy and thiol groups. Thesefunctional groups on the polymeric residues can be further reacted, ifdesired, with materials that are generally reactive with such functionalgroups and which can assist in targeting specific tissues in the bodyincluding, for example, diseased tissue. Exemplary materials which canbe reacted with the additional functional groups include, for example,proteins, including antibodies, carbohydrates, peptides, glycopeptides,glycolipids, lectins, and nucleosides.

In addition to residues of hydrophilic polymers, the chemical used toderivatize the polypeptides of the present invention can be a saccharideresidue. Exemplary saccharides which can be derived include, forexample, monosaccharides or sugar alcohols, such as erythrose, threose,ribose, arabinose, xylose, lyxose, fructose, sorbitol, mannitol andsedoheptulose, with preferred monosaccharides being fructose, mannose,xylose, arabinose, mannitol and sorbitol; and disaccharides, such aslactose, sucrose, maltose and cellobiose. Other saccharides include, forexample, inositol and ganglioside head groups. Other suitablesaccharides, in addition to those exemplified above, will be readilyapparent to one skilled in the art based on the present disclosure.Generally, saccharides which may be used for derivitization includesaccharides that can be attached to the polypeptides of the inventionvia alkylation or acylation reactions.

Moreover, the invention also encompasses derivitization of thepolypeptides of the present invention, for example, with lipids(including cationic, anionic, polymerized, charged, synthetic,saturated, unsaturated, and any combination of the above, etc.).stabilizing agents.

The invention encompasses derivitization of the polypeptides of thepresent invention, for example, with compounds that may serve astabilizing function (e.g., to increase the polypeptides half-life insolution, to make the polypeptides more water soluble, to increase thepolypeptides hydrophilic or hydrophobic character, etc.). Polymersuseful as stabilizing materials may be of natural, semi-synthetic(modified natural) or synthetic origin. Exemplary natural polymersinclude naturally occurring polysaccharides, such as, for example,arabinans, fructans, fucans, galactans, galacturonans, glucans, mannans,xylans (such as, for example, inulin), levan, fucoidan, carrageenan,galatocarolose, pectic acid, pectins, including amylose, pullulan,glycogen, amylopectin, cellulose, dextran, dextrin, dextrose, glucose,polyglucose, polydextrose, pustulan, chitin, agarose, keratin,chondroitin, dermatan, hyaluronic acid, alginic acid, xanthin gum,starch and various other natural homopolymer or heteropolymers, such asthose containing one or more of the following aldoses, ketoses, acids oramines: erythose, threose, ribose, arabinose, xylose, lyxose, allose,altrose, glucose, dextrose, mannose, gulose, idose, galactose, talose,erythrulose, ribulose, xylulose, psicose, fructose, sorbose, tagatose,mannitol, sorbitol, lactose, sucrose, trehalose, maltose, cellobiose,glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine,aspartic acid, glutamic acid, lysine, arginine, histidine, glucuronicacid, gluconic acid, glucaric acid, galacturonic acid, mannuronic acid,glucosamine, galactosamine, and neuraminic acid, and naturally occurringderivatives thereof Accordingly, suitable polymers include, for example,proteins, such as albumin, polyalginates, and polylactide-coglycolidepolymers. Exemplary semi-synthetic polymers includecarboxymethylcellulose, hydroxymethylcellulose,hydroxypropylmethylcellulose, methylcellulose, and methoxycellulose.Exemplary synthetic polymers include polyphosphazenes, hydroxyapatites,fluoroapatite polymers, polyethylenes (such as, for example,polyethylene glycol (including for example, the class of compoundsreferred to as Pluronics.RTM., commercially available from BASF,Parsippany, N.J.), polyoxyethylene, and polyethylene terephthlate),polypropylenes (such as, for example, polypropylene glycol),polyurethanes (such as, for example, polyvinyl alcohol (PVA), polyvinylchloride and polyvinylpyrrolidone), polyamides including nylon,polystyrene, polylactic acids, fluorinated hydrocarbon polymers,fluorinated carbon polymers (such as, for example,polytetrafluoroethylene), acrylate, methacrylate, andpolymethylmethacrylate, and derivatives thereof. Methods for thepreparation of derivatized polypeptides of the invention which employpolymers as stabilizing compounds will be readily apparent to oneskilled in the art, in view of the present disclosure, when coupled withinformation known in the art, such as that described and referred to inUnger, U.S. Pat. No. 5,205,290, the disclosure of which is herebyincorporated by reference herein in its entirety.

Moreover, the invention encompasses additional modifications of thepolypeptides of the present invention. Such additional modifications areknown in the art, and are specifically provided, in addition to methodsof derivitization, etc., in U.S. Pat. No. 6,028,066, which is herebyincorporated in its entirety herein.

The polypeptides of the invention may be in monomers or multimers (i.e.,dimers, trimers, tetramers and higher multimers). Accordingly, thepresent invention relates to monomers and multimers of the polypeptidesof the invention, their preparation, and compositions (preferably,Therapeutics) containing them. In specific embodiments, the polypeptidesof the invention are monomers, dimers, trimers or tetramers. Inadditional embodiments, the multimers of the invention are at leastdimers, at least trimers, or at least tetramers.

Multimers encompassed by the invention may be homomers or heteromers. Asused herein, the term homomer, refers to a multimer containing onlypolypeptides corresponding to the amino acid sequence of SEQ ID NO:2and/or 4 or encoded by the cDNA contained in a deposited clone(including fragments, variants, splice variants, and fusion proteins,corresponding to these polypeptides as described herein). These homomersmay contain polypeptides having identical or different amino acidsequences. In a specific embodiment, a homomer of the invention is amultimer containing only polypeptides having an identical amino acidsequence. In another specific embodiment, a homomer of the invention isa multimer containing polypeptides having different amino acidsequences. In specific embodiments, the multimer of the invention is ahomodimer (e.g., containing polypeptides having identical or differentamino acid sequences) or a homotrimer (e.g., containing polypeptideshaving identical and/or different amino acid sequences). In additionalembodiments, the homomeric multimer of the invention is at least ahomodimer, at least a homotrimer, or at least a homotetramer.

As used herein, the term heteromer refers to a multimer containing oneor more heterologous polypeptides (i.e., polypeptides of differentproteins) in addition to the polypeptides of the invention. In aspecific embodiment, the multimer of the invention is a heterodimer, aheterotrimer, or a heterotetramer. In additional embodiments, theheteromeric multimer of the invention is at least a heterodimer, atleast a heterotrimer, or at least a heterotetramer.

Multimers of the invention may be the result of hydrophobic,hydrophilic, ionic and/or covalent associations and/or may be indirectlylinked, by for example, liposome formation. Thus, in one embodiment,multimers of the invention, such as, for example, homodimers orhomotrimers, are formed when polypeptides of the invention contact oneanother in solution. In another embodiment, heteromultimers of theinvention, such as, for example, heterotrimers or heterotetramers, areformed when polypeptides of the invention contact antibodies to thepolypeptides of the invention (including antibodies to the heterologouspolypeptide sequence in a fusion protein of the invention) in solution.In other embodiments, multimers of the invention are form ed by covalentassociations with and/or between the polypeptides of the invention. Suchcovalent associations may involve one or more amino acid residuescontained in the polypeptide sequence (e.g., that recited in thesequence listing, or contained in the polypeptide encoded by a depositedclone). In one instance, the covalent associations are cross-linkingbetween cysteine residues located within the polypeptide sequences whichinteract in the native (i.e., naturally occurring) polypeptide. Inanother instance, the covalent associations are the consequence ofchemical or recombinant manipulation. Alternatively, such covalentassociations may involve one or more amino acid residues contained inthe heterologous polypeptide sequence in a fusion protein of theinvention.

In one example, covalent associations are between the heterologoussequence contained in a fusion protein of the invention (see, e.g., U.S.Pat. No. 5,478,925). In a specific example, the covalent associationsare between the heterologous sequence contained in an Fc fusion proteinof the invention (as described herein). In another specific example,covalent associations of fusion proteins of the invention are betweenheterologous polypeptide sequence from another protein that is capableof forming covalently associated multimers, such as for example,osteoprotegerin (see, e.g., International Publication NO: WO 98/49305,the contents of which are herein incorporated by reference in itsentirety). In another embodiment, two or more polypeptides of theinvention are joined through peptide linkers. Examples include thosepeptide linkers described in U.S. Pat. No. 5,073,627 (herebyincorporated by reference). Proteins comprising multiple polypeptides ofthe invention separated by peptide linkers may be produced usingconventional recombinant DNA technology.

Another method for preparing multimer polypeptides of the inventioninvolves use of polypeptides of the invention fused to a leucine zipperor isoleucine zipper polypeptide sequence. Leucine zipper and isoleucinezipper domains are polypeptides that promote multimerization of theproteins in which they are found. Leucine zippers were originallyidentified in several DNA-binding proteins (Landschulz et al., Science240:1759, (1988)), and have since been found in a variety of differentproteins. Among the known leucine zippers are naturally occurringpeptides and derivatives thereof that dimerize or trimerize. Examples ofleucine zipper domains suitable for producing soluble multimericproteins of the invention are those described in PCT application WO94/10308, hereby incorporated by reference. Recombinant fusion proteinscomprising a polypeptide of the invention fused to a polypeptidesequence that dimerizes or trimerizes in solution are expressed insuitable host cells, and the resulting soluble multimeric fusion proteinis recovered from the culture supernatant using techniques known in theart.

Trimeric polypeptides of the invention may offer the advantage ofenhanced biological activity. Preferred leucine zipper moieties andisoleucine moieties are those that preferentially form trimers. Oneexample is a leucine zipper derived from lung surfactant protein D(SPD), as described in Hoppe et al. (FEBS Letters 344:191, (1994)) andin U.S. patent application Ser. No. 08/446,922, hereby incorporated byreference. Other peptides derived from naturally occurring trimericproteins may be employed in preparing trimeric polypeptides of theinvention.

In another example, proteins of the invention are associated byinteractions between Flag® polypeptide sequence contained in fusionproteins of the invention containing Flag® polypeptide sequence. In afurther embodiment, associations proteins of the invention areassociated by interactions between heterologous polypeptide sequencecontained in Flag® fusion proteins of the invention and anti-Flag®antibody.

The multimers of the invention may be generated using chemicaltechniques known in the art. For example, polypeptides desired to becontained in the multimers of the invention may be chemicallycross-linked using linker molecules and linker molecule lengthoptimization techniques known in the art (see, e.g., U.S. Pat. No.5,478,925, which is herein incorporated by reference in its entirety).Additionally, multimers of the invention may be generated usingtechniques known in the art to form one or more inter-moleculecross-links between the cysteine residues located within the sequence ofthe polypeptides desired to be contained in the multimer (see, e.g.,U.S. Pat. No. 5,478,925, which is herein incorporated by reference inits entirety). Further, polypeptides of the invention may be routinelymodified by the addition of cysteine or biotin to the C terminus orN-terminus of the polypeptide and techniques known in the art may beapplied to generate multimers containing one or more of these modifiedpolypeptides (see, e.g., U.S. Pat. No. 5,478,925, which is hereinincorporated by reference in its entirety). Additionally, techniquesknown in the art may be applied to generate liposomes containing thepolypeptide components desired to be contained in the multimer of theinvention (see, e.g., U.S. Pat. No. 5,478,925, which is hereinincorporated by reference in its entirety).

Alternatively, multimers of the invention may be generated using geneticengineering techniques known in the art. In one embodiment, polypeptidescontained in multimers of the invention are produced recombinantly usingfusion protein technology described herein or otherwise known in the art(see, e.g., U.S. Pat. No. 5,478,925, which is herein incorporated byreference in its entirety). In a specific embodiment, polynucleotidescoding for a homodimer of the invention are generated by ligating apolynucleotide sequence encoding a polypeptide of the invention to asequence encoding a linker polypeptide and then further to a syntheticpolynucleotide encoding the translated product of the polypeptide in thereverse orientation from the original C-terminus to the N-terminus(lacking the leader sequence) (see, e.g., U.S. Pat. No. 5,478,925, whichis herein incorporated by reference in its entirety). In anotherembodiment, recombinant techniques described herein or otherwise knownin the art are applied to generate recombinant polypeptides of theinvention which contain a transmembrane domain (or hydrophobic or signalpeptide) and which can be incorporated by membrane reconstitutiontechniques into liposomes (see, e.g., U.S. Pat. No. 5,478,925, which isherein incorporated by reference in its entirety).

In addition, the polynucleotide insert of the present invention could beoperatively linked to “artificial” or chimeric promoters andtranscription factors. Specifically, the artificial promoter couldcomprise, or alternatively consist, of any combination of cis-acting DNAsequence elements that are recognized by trans-acting transcriptionfactors. Preferably, the cis acting DNA sequence elements andtrans-acting transcription factors are operable in mammals. Further, thetrans-acting transcription factors of such “artificial” promoters couldalso be “artificial” or chimeric in design themselves and could act asactivators or repressors to said “artificial” promoter.

Uses of the Polynucleotides

Each of the polynucleotides identified herein can be used in numerousways as reagents. The following description should be consideredexemplary and utilizes known techniques.

The polynucleotides of the present invention are useful for chromosomeidentification. There exists an ongoing need to identify new chromosomemarkers, since few chromosome marking reagents, based on actual sequencedata (repeat polymorphisms), are presently available. Eachpolynucleotide of the present invention can be used as a chromosomemarker.

Briefly, sequences can be mapped to chromosomes by preparing PCR primers(preferably 15-25 bp) from the sequences shown in SEQ ID NO:1 and/or 3.Primers can be selected using computer analysis so that primers do notspan more than one predicted exon in the genomic DNA. These primers arethen used for PCR screening of somatic cell hybrids containingindividual human chromosomes. Only those hybrids containing the humangene corresponding to the SEQ ID NO:1 and/or 3 will yield an amplifiedfragment.

Briefly, sequences can be mapped to chromosomes by preparing PCR primers(preferably 15-25 bp) from the sequences shown in SEQ ID NO:3. Primerscan be selected using computer analysis so that primers do not span morethan one predicted exon in the genomic DNA. These primers are then usedfor PCR screening of somatic cell hybrids containing individual humanchromosomes. Only those hybrids containing the human gene correspondingto the SEQ ID NO:3 will yield an amplified fragment.

Similarly, somatic hybrids provide a rapid method of PCR mapping thepolynucleotides to particular chromosomes. Three or more clones can beassigned per day using a single thermal cycler. Moreover,sublocalization of the polynucleotides can be achieved with panels ofspecific chromosome fragments. Other gene mapping strategies that can beused include in situ hybridization, prescreening with labeledflow-sorted chromosomes, and preselection by hybridization to constructchromosome specific-cDNA libraries.

Precise chromosomal location of the polynucleotides can also be achievedusing fluorescence in situ hybridization (FISH) of a metaphasechromosomal spread. This technique uses polynucleotides as short as 500or 600 bases; however, polynucleotides 2,000-4,000 bp are preferred. Fora review of this technique, see Verma et al., “Human Chromosomes: aManual of Basic Techniques,” Pergamon Press, New York (1988).

For chromosome mapping, the polynucleotides can be used individually (tomark a single chromosome or a single site on that chromosome) or inpanels (for marking multiple sites and/or multiple chromosomes).Preferred polynucleotides correspond to the noncoding regions of thecDNAs because the coding sequences are more likely conserved within genefamilies, thus increasing the chance of cross hybridization duringchromosomal mapping.

Once a polynucleotide has been mapped to a precise chromosomal location,the physical position of the polynucleotide can be used in linkageanalysis. Linkage analysis establishes coinheritance between achromosomal location and presentation of a particular disease. Diseasemapping data are known in the art. Assuming 1 megabase mappingresolution and one gene per 20 kb, a cDNA precisely localized to achromosomal region associated with the disease could be one of 50-500potential causative genes.

Thus, once coinheritance is established, differences in thepolynucleotide and the corresponding gene between affected andunaffected organisms can be examined. First, visible structuralalterations in the chromosomes, such as deletions or translocations, areexamined in chromosome spreads or by PCR. If no structural alterationsexist, the presence of point mutations are ascertained. Mutationsobserved in some or all affected organisms, but not in normal organisms,indicates that the mutation may cause the disease. However, completesequencing of the polypeptide and the corresponding gene from severalnormal organisms is required to distinguish the mutation from apolymorphism. If a new polymorphism is identified, this polymorphicpolypeptide can be used for further linkage analysis.

Furthermore, increased or decreased expression of the gene in affectedorganisms as compared to unaffected organisms can be assessed usingpolynucleotides of the present invention. Any of these alterations(altered expression, chromosomal rearrangement, or mutation) can be usedas a diagnostic or prognostic marker.

Thus, the invention also provides a diagnostic method useful duringdiagnosis of a disorder, involving measuring the expression level ofpolynucleotides of the present invention in cells or body fluid from anorganism and comparing the measured gene expression level with astandard level of polynucleotide expression level, whereby an increaseor decrease in the gene expression level compared to the standard isindicative of a disorder.

By “measuring the expression level of a polynucleotide of the presentinvention” is intended qualitatively or quantitatively measuring orestimating the level of the polypeptide of the present invention or thelevel of the mRNA encoding the polypeptide in a first biological sampleeither directly (e.g., by determining or estimating absolute proteinlevel or mRNA level) or relatively (e.g., by comparing to thepolypeptide level or mRNA level in a second biological sample).Preferably, the polypeptide level or mRNA level in the first biologicalsample is measured or estimated and compared to a standard polypeptidelevel or mRNA level, the standard being taken from a second biologicalsample obtained from an individual not having the disorder or beingdetermined by averaging levels from a population of organisms not havinga disorder. As will be appreciated in the art, once a standardpolypeptide level or mRNA level is known, it can be used repeatedly as astandard for comparison.

By “biological sample” is intended any biological sample obtained froman organism, body fluids, cell line, tissue culture, or other sourcewhich contains the polypeptide of the present invention or mRNA. Asindicated, biological samples include body fluids (such as the followingnon-limiting examples, sputum, amniotic fluid, urine, saliva, breastmilk, secretions, interstitial fluid, blood, serum, spinal fluid, etc.)which contain the polypeptide of the present invention, and other tissuesources found to express the polypeptide of the present invention.Methods for obtaining tissue biopsies and body fluids from organisms arewell known in the art. Where the biological sample is to include mRNA, atissue biopsy is the preferred source.

The method(s) provided above may Preferably be applied in a diagnosticmethod and/or kits in which polynucleotides and/or polypeptides areattached to a solid support. In one exemplary method, the support may bea “gene chip” or a “biological chip” as described in U.S. Pat. Nos.5,837,832, 5,874,219, and 5,856,174. Further, such a gene chip withpolynucleotides of the present invention attached may be used toidentify polymorphisms between the polynucleotide sequences, withpolynucleotides isolated from a test subject. The knowledge of suchpolymorphisms (i.e. their location, as well as, their existence) wouldbe beneficial in identifying disease loci for many disorders, includingproliferative diseases and conditions. Such a method is described inU.S. Pat. Nos. 5,858,659 and 5,856,104. The US Patents referenced supraare hereby incorporated by reference in their entirety herein.

The present invention encompasses polynucleotides of the presentinvention that are chemically synthesized, or reproduced as peptidenucleic acids (PNA), or according to other methods known in the art. Theuse of PNAs would serve as the preferred form if the polynucleotides areincorporated onto a solid support, or gene chip. For the purposes of thepresent invention, a peptide nucleic acid (PNA) is a polyamide type ofDNA analog and the monomeric units for adenine, guanine, thymine andcytosine are available commercially (Perceptive Biosystems). Certaincomponents of DNA, such as phosphorus, phosphorus oxides, or deoxyribosederivatives, are not present in PNAs. As disclosed by P. E. Nielsen, M.Egholm, R. H. Berg and O. Buchardt, Science 254, 1497 (1991); and M.Egholm, O. Buchardt, L. Christensen, C. Behrens, S. M. Freier, D. A.Driver, R. H. Berg, S. K. Kim, B. Norden, and P. E. Nielsen, Nature 365,666 (1993), PNAs bind specifically and tightly to complementary DNAstrands and are not degraded by nucleases. In fact, PNA binds morestrongly to DNA than DNA itself does. This is probably because there isno electrostatic repulsion between the two strands, and also thepolyamide backbone is more flexible. Because of this, PNA/DNA duplexesbind under a wider range of stringency conditions than DNA/DNA duplexes,making it easier to perform multiplex hybridization. Smaller probes canbe used than with DNA due to the stronger binding characteristics ofPNA:DNA hybrids. In addition, it is more likely that single basemismatches can be determined with PNA/DNA hybridization because a singlemismatch in a PNA/DNA 15-mer lowers the melting point (T.sub.m) by8°-20° C., vs. 4°-16° C. for the DNA/DNA 15-mer duplex. Also, theabsence of charge groups in PNA means that hybridization can be done atlow ionic strengths and reduce possible interference by salt during theanalysis.

In addition to the foregoing, a polynucleotide can be used to controlgene expression through triple helix formation or antisense DNA or RNA.Antisense techniques are discussed, for example, in Okano, J. Neurochem.56: 560 (1991); “Oligodeoxynucleotides as Antisense Inhibitors of GeneExpression,” CRC Press, Boca Raton, Fla. (1988). Triple helix formationis discussed in, for instance Lee et al., Nucleic Acids Research 6: 3073(1979); Cooney et al., Science 241: 456 (1988); and Dervan et al.,Science 251: 1360 (1991). Both methods rely on binding of thepolynucleotide to a complementary DNA or RNA. For these techniques,preferred polynucleotides are usually oligonucleotides 20 to 40 bases inlength and complementary to either the region of the gene involved intranscription (triple helix—see Lee et al., Nucl. Acids Res. 6:3073(1979); Cooney et al., Science 241:456 (1988); and Dervan et al.,Science 251:1360 (1991) ) or to the mRNA itself (antisense—Okano, J.Neurochem. 56:560 (1991); Oligodeoxy-nucleotides as Antisense Inhibitorsof Gene Expression, CRC Press, Boca Raton, Fla. (1988).) Triple helixformation optimally results in a shut-off of RNA transcription from DNA,while antisense RNA hybridization blocks translation of an mRNA moleculeinto polypeptide. Both techniques are effective in model systems, andthe information disclosed herein can be used to design antisense ortriple helix polynucleotides in an effort to treat or prevent disease.

The present invention encompasses the addition of a nuclear localizationsignal, operably linked to the 5′ end, 3′ end, or any location therein,to any of the oligonucleotides, antisense oligonucleotides, triple helixoligonucleotides, ribozymes, PNA oligonucleotides, and/orpolynucleotides, of the present invention. See, for example, G. Cutrona,et al., Nat. Biotech., 18:300-303, (2000); which is hereby incorporatedherein by reference.

Polynucleotides of the present invention are also useful in genetherapy. One goal of gene therapy is to insert a normal gene into anorganism having a defective gene, in an effort to correct the geneticdefect. The polynucleotides disclosed in the present invention offer ameans of targeting such genetic defects in a highly accurate manner.Another goal is to insert a new gene that was not present in the hostgenome, thereby producing a new trait in the host cell. In one example,polynucleotide sequences of the present invention may be used toconstruct chimeric RNA/DNA oligonucleotides corresponding to saidsequences, specifically designed to induce host cell mismatch repairmechanisms in an organism upon systemic injection, for example(Bartlett, R. J., et al., Nat. Biotech, 18:615-622 (2000), which ishereby incorporated by reference herein in its entirety). Such RNA/DNAoligonucleotides could be designed to correct genetic defects in certainhost strains, and/or to introduce desired phenotypes in the host (e.g.,introduction of a specific polymorphism within an endogenous genecorresponding to a polynucleotide of the present invention that mayameliorate and/or prevent a disease symptom and/or disorder, etc.).Alternatively, the polynucleotide sequence of the present invention maybe used to construct duplex oligonucleotides corresponding to saidsequence, specifically designed to correct genetic defects in certainhost strains, and/or to introduce desired phenotypes into the host(e.g., introduction of a specific polymorphism within an endogenous genecorresponding to a polynucleotide of the present invention that mayameliorate and/or prevent a disease symptom and/or disorder, etc). Suchmethods of using duplex oligonucleotides are known in the art and areencompassed by the present invention (see EP1007712, which is herebyincorporated by reference herein in its entirety).

The polynucleotides are also useful for identifying organisms fromminute biological samples. The United States military, for example, isconsidering the use of restriction fragment length polymorphism (RFLP)for identification of its personnel. In this technique, an individual'sgenomic DNA is digested with one or more restriction enzymes, and probedon a Southern blot to yield unique bands for identifying personnel. Thismethod does not suffer from the current limitations of “Dog Tags” whichcan be lost, switched, or stolen, making positive identificationdifficult. The polynucleotides of the present invention can be used asadditional DNA markers for RFLP.

The polynucleotides of the present invention can also be used as analternative to RFLP, by determining the actual base-by-base DNA sequenceof selected portions of an organisms genome. These sequences can be usedto prepare PCR primers for amplifying and isolating such selected DNA,which can then be sequenced. Using this technique, organisms can beidentified because each organism will have a unique set of DNAsequences. Once an unique ID database is established for an organism,positive identification of that organism, living or dead, can be madefrom extremely small tissue samples. Similarly, polynucleotides of thepresent invention can be used as polymorphic markers, in addition to,the identification of transformed or non-transformed cells and/ortissues.

There is also a need for reagents capable of identifying the source of aparticular tissue. Such need arises, for example, when presented withtissue of unknown origin. Appropriate reagents can comprise, forexample, DNA probes or primers specific to particular tissue preparedfrom the sequences of the present invention. Panels of such reagents canidentify tissue by species and/or by organ type. In a similar fashion,these reagents can be used to screen tissue cultures for contamination.Moreover, as mentioned above, such reagents can be used to screen and/oridentify transformed and non-transformed cells and/or tissues.

In the very least, the polynucleotides of the present invention can beused as molecular weight markers on Southern gels, as diagnostic probesfor the presence of a specific mRNA in a particular cell type, as aprobe to “subtract-out” known sequences in the process of discoveringnovel polynucleotides, for selecting and making oligomers for attachmentto a “gene chip” or other support, to raise anti-DNA antibodies usingDNA immunization techniques, and as an antigen to elicit an immuneresponse.

Uses of the Polypeptides

Each of the polypeptides identified herein can be used in numerous ways.The following description should be considered exemplary and utilizesknown techniques.

A polypeptide of the present invention can be used to assay proteinlevels in a biological sample using antibody-based techniques. Forexample, protein expression in tissues can be studied with classicalimmunohistological methods. (Jalkanen, M., et al., J. Cell. Biol.101:976-985 (1985); Jalkanen, M., et al., J. Cell . Biol. 105:3087-3096(1987).) Other antibody-based methods useful for detecting protein geneexpression include immunoassays, such as the enzyme linked immunosorbentassay (ELISA) and the radioimmunoassay (RIA). Suitable antibody assaylabels are known in the art and include enzyme labels, such as, glucoseoxidase, and radioisotopes, such as iodine (125I, 121I), carbon (14C),sulfur (35S), tritium (3H), indium (112In), and technetium (99mTc), andfluorescent labels, such as fluorescein and rhodamine, and biotin.

In addition to assaying protein levels in a biological sample, proteinscan also be detected in vivo by imaging. Antibody labels or markers forin vivo imaging of protein include those detectable by X-radiography,NMR or ESR. For X-radiography, suitable labels include radioisotopessuch as barium or cesium, which emit detectable radiation but are notovertly harmful to the subject. Suitable markers for NMR and ESR includethose with a detectable characteristic spin, such as deuterium, whichmay be incorporated into the antibody by labeling of nutrients for therelevant hybridoma.

A protein-specific antibody or antibody fragment which has been labeledwith an appropriate detectable imaging moiety, such as a radioisotope(for example, 131I, 112In, 99mTc), a radio-opaque substance, or amaterial detectable by nuclear magnetic resonance, is introduced (forexample, parenterally, subcutaneously, or intraperitoneally) into themammal. It will be understood in the art that the size of the subjectand the imaging system used will determine the quantity of imagingmoiety needed to produce diagnostic images. In the case of aradioisotope moiety, for a human subject, the quantity of radioactivityinjected will normally range from about 5 to 20 millicuries of 99mTc.The labeled antibody or antibody fragment will then preferentiallyaccumulate at the location of cells which contain the specific protein.In vivo tumor imaging is described in S. W. Burchiel et al.,“Immunopharmacokinetics of Radiolabeled Antibodies and Their Fragments.”(Chapter 13 in Tumor Imaging: The Radiochemical Detection of Cancer, S.W. Burchiel and B. A. Rhodes, eds., Masson Publishing Inc. (1982).)

Thus, the invention provides a diagnostic method of a disorder, whichinvolves (a) assaying the expression of a polypeptide of the presentinvention in cells or body fluid of an individual; (b) comparing thelevel of gene expression with a standard gene expression level, wherebyan increase or decrease in the assayed polypeptide gene expression levelcompared to the standard expression level is indicative of a disorder.With respect to cancer, the presence of a relatively high amount oftranscript in biopsied tissue from an individual may indicate apredisposition for the development of the disease, or may provide ameans for detecting the disease prior to the appearance of actualclinical symptoms. A more definitive diagnosis of this type may allowhealth professionals to employ preventative measures or aggressivetreatment earlier thereby preventing the development or furtherprogression of the cancer.

Moreover, polypeptides of the present invention can be used to treat,prevent, and/or diagnose disease. For example, patients can beadministered a polypeptide of the present invention in an effort toreplace absent or decreased levels of the polypeptide (e.g., insulin),to supplement absent or decreased levels of a different polypeptide(e.g., hemoglobin S for hemoglobin B, SOD, catalase, DNA repairproteins), to inhibit the activity of a polypeptide (e.g., an oncogeneor tumor suppressor), to activate the activity of a polypeptide (e.g.,by binding to a receptor), to reduce the activity of a membrane boundreceptor by competing with it for free ligand (e.g., soluble TNFreceptors used in reducing inflammation), or to bring about a desiredresponse (e.g., blood vessel growth inhibition, enhancement of theimmune response to proliferative cells or tissues).

Similarly, antibodies directed to a polypeptide of the present inventioncan also be used to treat, prevent, and/or diagnose disease. Forexample, administration of an antibody directed to a polypeptide of thepresent invention can bind and reduce overproduction of the polypeptide.Similarly, administration of an antibody can activate the polypeptide,such as by binding to a polypeptide bound to a membrane (receptor).

At the very least, the polypeptides of the present invention can be usedas molecular weight markers on SDS-PAGE gels or on molecular sieve gelfiltration columns using methods well known to those of skill in theart. Polypeptides can also be used to raise antibodies, which in turnare used to measure protein expression from a recombinant cell, as a wayof assessing transformation of the host cell. Moreover, the polypeptidesof the present invention can be used to test the following biologicalactivities.

Gene Therapy Methods

Another aspect of the present invention is to gene therapy methods fortreating or preventing disorders, diseases and conditions. The genetherapy methods relate to the introduction of nucleic acid (DNA, RNA andantisense DNA or RNA) sequences into an animal to achieve expression ofa polypeptide of the present invention. This method requires apolynucleotide which codes for a polypeptide of the invention thatoperatively linked to a promoter and any other genetic elementsnecessary for the expression of the polypeptide by the target tissue.Such gene therapy and delivery techniques are known in the art, see, forexample, WO90/11092, which is herein incorporated by reference.

Thus, for example, cells from a patient may be engineered with apolynucleotide (DNA or RNA) comprising a promoter operably linked to apolynucleotide of the invention ex vivo, with the engineered cells thenbeing provided to a patient to be treated with the polypeptide. Suchmethods are well-known in the art. For example, see Belldegrun et al.,J. Natl. Cancer Inst., 85:207-216 (1993); Ferrantini et al., CancerResearch, 53:107-1112 (1993); Ferrantini et al., J. Immunology 153:4604-4615 (1994); Kaido, T., et al., Int. J. Cancer 60: 221-229 (1995);Ogura et al., Cancer Research 50: 5102-5106 (1990); Santodonato, et al.,Human Gene Therapy 7:1-10 (1996); Santodonato, et al., Gene Therapy4:1246-1255 (1997); and Zhang, et al., Cancer Gene Therapy 3: 31-38(1996)), which are herein incorporated by reference. In one embodiment,the cells which are engineered are arterial cells. The arterial cellsmay be reintroduced into the patient through direct injection to theartery, the tissues surrounding the artery, or through catheterinjection.

As discussed in more detail below, the polynucleotide constructs can bedelivered by any method that delivers injectable materials to the cellsof an animal, such as, injection into the interstitial space of tissues(heart, muscle, skin, lung, liver, and the like). The polynucleotideconstructs may be delivered in a pharmaceutically acceptable liquid oraqueous carrier.

In one embodiment, the polynucleotide of the invention is delivered as anaked polynucleotide. The term “naked” polynucleotide, DNA or RNA refersto sequences that are free from any delivery vehicle that acts toassist, promote or facilitate entry into the cell, including viralsequences, viral particles, liposome formulations, lipofectin orprecipitating agents and the like. However, the polynucleotides of theinvention can also be delivered in liposome formulations and lipofectinformulations and the like can be prepared by methods well known to thoseskilled in the art. Such methods are described, for example, in U.S.Pat. Nos. 5,593,972, 5,589,466, and 5,580,859, which are hereinincorporated by reference.

The polynucleotide vector constructs of the invention used in the genetherapy method are preferably constructs that will not integrate intothe host genome nor will they contain sequences that allow forreplication. Appropriate vectors include pWLNEO, pSV2CAT, pOG44, pXT1and pSG available from Stratagene; pSVK3, pBPV, pMSG and pSVL availablefrom Pharmacia; and pEF1/V5, pcDNA3.1, and pRc/CMV2 available fromInvitrogen. Other suitable vectors will be readily apparent to theskilled artisan.

Any strong promoter known to those skilled in the art can be used fordriving the expression of polynucleotide sequence of the invention.Suitable promoters include adenoviral promoters, such as the adenoviralmajor late promoter; or heterologous promoters, such as thecytomegalovirus (CMV) promoter; the respiratory syncytial virus (RSV)promoter; inducible promoters, such as the MMT promoter, themetallothionein promoter; heat shock promoters; the albumin promoter;the ApoAI promoter; human globin promoters; viral thymidine kinasepromoters, such as the Herpes Simplex thymidine kinase promoter;retroviral LTRs; the b-actin promoter; and human growth hormonepromoters. The promoter also may be the native promoter for thepolynucleotides of the invention.

Unlike other gene therapy techniques, one major advantage of introducingnaked nucleic acid sequences into target cells is the transitory natureof the polynucleotide synthesis in the cells. Studies have shown thatnon-replicating DNA sequences can be introduced into cells to provideproduction of the desired polypeptide for periods of up to six months.

The polynucleotide construct of the invention can be delivered to theinterstitial space of tissues within the an animal, including of muscle,skin, brain, lung, liver, spleen, bone marrow, thymus, heart, lymph,blood, bone, cartilage, pancreas, kidney, gall bladder, stomach,intestine, testis, ovary, uterus, rectum, nervous system, eye, gland,and connective tissue. Interstitial space of the tissues comprises theintercellular, fluid, mucopolysaccharide matrix among the reticularfibers of organ tissues, elastic fibers in the walls of vessels orchambers, collagen fibers of fibrous tissues, or that same matrix withinconnective tissue ensheathing muscle cells or in the lacunae of bone. Itis similarly the space occupied by the plasma of the circulation and thelymph fluid of the lymphatic channels. Delivery to the interstitialspace of muscle tissue is preferred for the reasons discussed below.They may be conveniently delivered by injection into the tissuescomprising these cells. They are preferably delivered to and expressedin persistent, non-dividing cells which are differentiated, althoughdelivery and expression may be achieved in non-differentiated or lesscompletely differentiated cells, such as, for example, stem cells ofblood or skin fibroblasts. In vivo muscle cells are particularlycompetent in their ability to take up and express polynucleotides.

For the naked nucleic acid sequence injection, an effective dosageamount of DNA or RNA will be in the range of from about 0.05 mg/kg bodyweight to about 50 mg/kg body weight. Preferably the dosage will be fromabout 0.005 mg/kg to about 20 mg/kg and more preferably from about 0.05mg/kg to about 5 mg/kg. Of course, as the artisan of ordinary skill willappreciate, this dosage will vary according to the tissue site ofinjection. The appropriate and effective dosage of nucleic acid sequencecan readily be determined by those of ordinary skill in the art and maydepend on the condition being treated and the route of administration.

The preferred route of administration is by the parenteral route ofinjection into the interstitial space of tissues. However, otherparenteral routes may also be used, such as, inhalation of an aerosolformulation particularly for delivery to lungs or bronchial tissues,throat or mucous membranes of the nose. In addition, naked DNAconstructs can be delivered to arteries during angioplasty by thecatheter used in the procedure.

The naked polynucleotides are delivered by any method known in the art,including, but not limited to, direct needle injection at the deliverysite, intravenous injection, topical administration, catheter infusion,and so-called “gene guns”. These delivery methods are known in the art.

The constructs may also be delivered with delivery vehicles such asviral sequences, viral particles, liposome formulations, lipofectin,precipitating agents, etc. Such methods of delivery are known in theart.

In certain embodiments, the polynucleotide constructs of the inventionare complexed in a liposome preparation. Liposomal preparations for usein the instant invention include cationic (positively charged), anionic(negatively charged) and neutral preparations. However, cationicliposomes are particularly preferred because a tight charge complex canbe formed between the cationic liposome and the polyanionic nucleicacid. Cationic liposomes have been shown to mediate intracellulardelivery of plasmid DNA (Felgner et al., Proc. Natl. Acad. Sci. USA,84:7413-7416 (1987), which is herein incorporated by reference); mRNA(Malone et al., Proc. Natl. Acad. Sci. USA, 86:6077-6081 (1989), whichis herein incorporated by reference); and purified transcription factors(Debs et al., J. Biol. Chem., 265:10189-10192 (1990), which is hereinincorporated by reference), in functional form.

Cationic liposomes are readily available. For example,N[1-2,3-dioleyloxy)propyl]-N,N,N-triethylammonium (DOTMA) liposomes areparticularly useful and are available under the trademark Lipofectin,from GIBCO BRL, Grand Island, N.Y. (See, also, Felgner et al., Proc.Natl. Acad. Sci. USA, 84:7413-7416 (1987), which is herein incorporatedby reference). Other commercially available liposomes includetransfectace (DDAB/DOPE) and DOTAP/DOPE (Boehringer).

Other cationic liposomes can be prepared from readily availablematerials using techniques well known in the art. See, e.g. PCTPublication NO: WO 90/11092 (which is herein incorporated by reference)for a description of the synthesis of DOTAP(1,2-bis(oleoyloxy)-3-(trimethylammonio)propane) liposomes. Preparationof DOTMA liposomes is explained in the literature, see, e.g., Felgner etal., Proc. Natl. Acad. Sci. USA, 84:7413-7417, which is hereinincorporated by reference. Similar methods can be used to prepareliposomes from other cationic lipid materials.

Similarly, anionic and neutral liposomes are readily available, such asfrom Avanti Polar Lipids (Birmingham, Ala.), or can be easily preparedusing readily available materials. Such materials include phosphatidyl,choline, cholesterol, phosphatidyl ethanolamine, dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidyl glycerol (DOPG),dioleoylphoshatidyl ethanolamine (DOPE), among others. These materialscan also be mixed with the DOTMA and DOTAP starting materials inappropriate ratios. Methods for making liposomes using these materialsare well known in the art.

For example, commercially dioleoylphosphatidyl choline (DOPC),dioleoylphosphatidyl glycerol (DOPG), and dioleoylphosphatidylethanolamine (DOPE) can be used in various combinations to makeconventional liposomes, with or without the addition of cholesterol.Thus, for example, DOPG/DOPC vesicles can be prepared by drying 50 mgeach of DOPG and DOPC under a stream of nitrogen gas into a sonicationvial. The sample is placed under a vacuum pump overnight and is hydratedthe following day with deionized water. The sample is then sonicated for2 hours in a capped vial, using a Heat Systems model 350 sonicatorequipped with an inverted cup (bath type) probe at the maximum settingwhile the bath is circulated at 15EC. Alternatively, negatively chargedvesicles can be prepared without sonication to produce multilamellarvesicles or by extrusion through nucleopore membranes to produceunilamellar vesicles of discrete size. Other methods are known andavailable to those of skill in the art.

The liposomes can comprise multilamellar vesicles (MLVs), smallunilamellar vesicles (SUVs), or large unilamellar vesicles (LUVs), withSUVs being preferred. The various liposome-nucleic acid complexes areprepared using methods well known in the art. See, e.g., Straubinger etal., Methods of Immunology, 101:512-527 (1983), which is hereinincorporated by reference. For example, MLVs containing nucleic acid canbe prepared by depositing a thin film of phospholipid on the walls of aglass tube and subsequently hydrating with a solution of the material tobe encapsulated. SUVs are prepared by extended sonication of MLVs toproduce a homogeneous population of unilamellar liposomes. The materialto be entrapped is added to a suspension of preformed MLVs and thensonicated. When using liposomes containing cationic lipids, the driedlipid film is resuspended in an appropriate solution such as sterilewater or an isotonic buffer solution such as 10 mM Tris/NaCl, sonicated,and then the preformed liposomes are mixed directly with the DNA. Theliposome and DNA form a very stable complex due to binding of thepositively charged liposomes to the cationic DNA. SUVs find use withsmall nucleic acid fragments. LUVs are prepared by a number of methods,well known in the art. Commonly used methods include Ca2+-EDTA chelation(Papahadjopoulos et al., Biochim. Biophys. Acta, 394:483 (1975); Wilsonet al., Cell, 17:77 (1979)); ether injection (Deamer et al., Biochim.Biophys. Acta, 443:629 (1976); Ostro et al., Biochem. Biophys. Res.Commun., 76:836 (1977); Fraley et al., Proc. Natl. Acad. Sci. USA,76:3348 (1979)); detergent dialysis (Enoch et al., Proc. Natl. Acad.Sci. USA, 76:145 (1979)); and reverse-phase evaporation (REV) (Fraley etal., J. Biol. Chem., 255:10431 (1980); Szoka et al., Proc. Natl. Acad.Sci. USA, 75:145 (1978); Schaefer-Ridder et al., Science, 215:166(1982)), which are herein incorporated by reference.

Generally, the ratio of DNA to liposomes will be from about 10:1 toabout 1:10. Preferably, the ration will be from about 5:1 to about 1:5.More preferably, the ration will be about 3:1 to about 1:3. Still morepreferably, the ratio will be about 1:1.

U.S. Pat. No. 5,676,954 (which is herein incorporated by reference)reports on the injection of genetic material, complexed with cationicliposomes carriers, into mice. U.S. Pat. Nos. 4,897,355, 4,946,787,5,049,386, 5,459,127, 5,589,466, 5,693,622, 5,580,859, 5,703,055, andinternational publication NO: WO 94/9469 (which are herein incorporatedby reference) provide cationic lipids for use in transfecting DNA intocells and mammals. U.S. Pat. Nos. 5,589,466, 5,693,622, 5,580,859,5,703,055, and international publication NO: WO 94/9469 (which areherein incorporated by reference) provide methods for deliveringDNA-cationic lipid complexes to mammals.

In certain embodiments, cells are engineered, ex vivo or in vivo, usinga retroviral particle containing RNA which comprises a sequence encodingpolypeptides of the invention. Retroviruses from which the retroviralplasmid vectors may be derived include, but are not limited to, MoloneyMurine Leukemia Virus, spleen necrosis virus, Rous sarcoma Virus, HarveySarcoma Virus, avian leukosis virus, gibbon ape leukemia virus, humanimmunodeficiency virus, Myeloproliferative Sarcoma Virus, and mammarytumor virus.

The retroviral plasmid vector is employed to transduce packaging celllines to form producer cell lines. Examples of packaging cells which maybe transfected include, but are not limited to, the PE501, PA317, R-2,R-AM, PA12, T19-14X, VT-19-17-H2, RCRE, RCRIP, GP+E-86, GP+envAm12, andDAN cell lines as described in Miller, Human Gene Therapy , 1:5-14(1990), which is incorporated herein by reference in its entirety. Thevector may transduce the packaging cells through any means known in theart. Such means include, but are not limited to, electroporation, theuse of liposomes, and CaPO4 precipitation. In one alternative, theretroviral plasmid vector may be encapsulated into a liposome, orcoupled to a lipid, and then administered to a host.

The producer cell line generates infectious retroviral vector particleswhich include polynucleotide encoding polypeptides of the invention.Such retroviral vector particles then may be employed, to transduceeukaryotic cells, either in vitro or in vivo. The transduced eukaryoticcells will express polypeptides of the invention.

In certain other embodiments, cells are engineered, ex vivo or in vivo,with polynucleotides of the invention contained in an adenovirus vector.Adenovirus can be manipulated such that it encodes and expressespolypeptides of the invention, and at the same time is inactivated interms of its ability to replicate in a normal lytic viral life cycle.Adenovirus expression is achieved without integration of the viral DNAinto the host cell chromosome, thereby alleviating concerns aboutinsertional mutagenesis. Furthermore, adenoviruses have been used aslive enteric vaccines for many years with an excellent safety profile(Schwartzet al., Am. Rev. Respir. Dis., 109:233-238 (1974)). Finally,adenovirus mediated gene transfer has been demonstrated in a number ofinstances including transfer of alpha-1-antitrypsin and CFTR to thelungs of cotton rats (Rosenfeld et al., Science, 252:431-434 (1991);Rosenfeld et al., Cell, 68:143-155 (1992)). Furthermore, extensivestudies to attempt to establish adenovirus as a causative agent in humancancer were uniformly negative (Green et al. Proc. Natl. Acad. Sci. USA,76:6606 (1979)).

Suitable adenoviral vectors useful in the present invention aredescribed, for example, in Kozarsky and Wilson, Curr. Opin. Genet.Devel., 3:499-503 (1993); Rosenfeld et al., Cell, 68:143-155 (1992);Engelhardt et al., Human Genet. Ther., 4:759-769 (1993); Yang et al.,Nature Genet., 7:362-369 (1994); Wilson et al., Nature, 365:691-692(1993); and U.S. Pat. No. 5,652,224, which are herein incorporated byreference. For example, the adenovirus vector Ad2 is useful and can begrown in human 293 cells. These cells contain the E1 region ofadenovirus and constitutively express E1a and E1b, which complement thedefective adenoviruses by providing the products of the genes deletedfrom the vector. In addition to Ad2, other varieties of adenovirus(e.g., Ad3, Ad5, and Ad7) are also useful in the present invention.

Preferably, the adenoviruses used in the present invention arereplication deficient. Replication deficient adenoviruses require theaid of a helper virus and/or packaging cell line to form infectiousparticles. The resulting virus is capable of infecting cells and canexpress a polynucleotide of interest which is operably linked to apromoter, but cannot replicate in most cells. Replication deficientadenoviruses may be deleted in one or more of all or a portion of thefollowing genes: E1a, E1b, E3, E4, E2a, or L1 through L5.

In certain other embodiments, the cells are engineered, ex vivo or invivo, using an adeno-associated virus (AAV). AAVs are naturallyoccurring defective viruses that require helper viruses to produceinfectious particles (Muzyczka, Curr. Topics in Microbiol. Immunol.,158:97 (1992)). It is also one of the few viruses that may integrate itsDNA into non-dividing cells. Vectors containing as little as 300 basepairs of AAV can be packaged and can integrate, but space for exogenousDNA is limited to about 4.5 kb. Methods for producing and using suchAAVs are known in the art. See, for example, U.S. Pat. Nos. 5,139,941,5,173,414, 5,354,678, 5,436,146, 5,474,935, 5,478,745, and 5,589,377.

For example, an appropriate AAV vector for use in the present inventionwill include all the sequences necessary for DNA replication,encapsidation, and host-cell integration. The polynucleotide constructcontaining polynucleotides of the invention is inserted into the AAVvector using standard cloning methods, such as those found in Sambrooket al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press(1989). The recombinant AAV vector is then transfected into packagingcells which are infected with a helper virus, using any standardtechnique, including lipofection, electroporation, calcium phosphateprecipitation, etc. Appropriate helper viruses include adenoviruses,cytomegaloviruses, vaccinia viruses, or herpes viruses. Once thepackaging cells are transfected and infected, they will produceinfectious AAV viral particles which contain the polynucleotideconstruct of the invention. These viral particles are then used totransduce eukaryotic cells, either ex vivo or in vivo. The transducedcells will contain the polynucleotide construct integrated into itsgenome, and will express the desired gene product.

Another method of gene therapy involves operably associatingheterologous control regions and endogenous polynucleotide sequences(e.g. encoding the polypeptide sequence of interest) via homologousrecombination (see, e.g., U.S. Pat. No. 5,641,670, issued Jun. 24, 1997;International Publication NO: WO 96/29411, published Sep. 26, 1996;International Publication NO: WO 94/12650, published Aug. 4, 1994;Koller et al., Proc. Natl. Acad. Sci. USA, 86:8932-8935 (1989); andZijlstra et al., Nature, 342:435-438 (1989). This method involves theactivation of a gene which is present in the target cells, but which isnot normally expressed in the cells, or is expressed at a lower levelthan desired.

Polynucleotide constructs are made, using standard techniques known inthe art, which contain the promoter with targeting sequences flankingthe promoter. Suitable promoters are described herein. The targetingsequence is sufficiently complementary to an endogenous sequence topermit homologous recombination of the promoter-targeting sequence withthe endogenous sequence. The targeting sequence will be sufficientlynear the 5′ end of the desired endogenous polynucleotide sequence so thepromoter will be operably linked to the endogenous sequence uponhomologous recombination.

The promoter and the targeting sequences can be amplified using PCR.Preferably, the amplified promoter contains distinct restriction enzymesites on the 5′ and 3′ ends. Preferably, the 3′ end of the firsttargeting sequence contains the same restriction enzyme site as the 5′end of the amplified promoter and the 5′ end of the second targetingsequence contains the same restriction site as the 3′ end of theamplified promoter. The amplified promoter and targeting sequences aredigested and ligated together.

The promoter-targeting sequence construct is delivered to the cells,either as naked polynucleotide, or in conjunction withtransfection-facilitating agents, such as liposomes, viral sequences,viral particles, whole viruses, lipofection, precipitating agents, etc.,described in more detail above. The P promoter-targeting sequence can bedelivered by any method, included direct needle injection, intravenousinjection, topical administration, catheter infusion, particleaccelerators, etc. The methods are described in more detail below.

The promoter-targeting sequence construct is taken up by cells.Homologous recombination between the construct and the endogenoussequence takes place, such that an endogenous sequence is placed underthe control of the promoter. The promoter then drives the expression ofthe endogenous sequence.

The polynucleotides encoding polypeptides of the present invention maybe administered along with other polynucleotides encoding angiogenicproteins. Angiogenic proteins include, but are not limited to, acidicand basic fibroblast growth factors, VEGF-1, VEGF-2 (VEGF-C), VEGF-3(VEGF-B), epidermal growth factor alpha and beta, platelet-derivedendothelial cell growth factor, platelet-derived growth factor, tumornecrosis factor alpha, hepatocyte growth factor, insulin like growthfactor, colony stimulating factor, macrophage colony stimulating factor,granulocyte/macrophage colony stimulating factor, and nitric oxidesynthase.

Preferably, the polynucleotide encoding a polypeptide of the inventioncontains a secretory signal sequence that facilitates secretion of theprotein. Typically, the signal sequence is positioned in the codingregion of the polynucleotide to be expressed towards or at the 5′ end ofthe coding region. The signal sequence may be homologous or heterologousto the polynucleotide of interest and may be homologous or heterologousto the cells to be transfected. Additionally, the signal sequence may bechemically synthesized using methods known in the art.

Any mode of administration of any of the above-described polynucleotidesconstructs can be used so long as the mode results in the expression ofone or more molecules in an amount sufficient to provide a therapeuticeffect. This includes direct needle injection, systemic injection,catheter infusion, biolistic injectors, particle accelerators (i.e.,“gene guns”), gelfoam sponge depots, other commercially available depotmaterials, osmotic pumps (e.g., Alza minipumps), oral or suppositorialsolid (tablet or pill) pharmaceutical formulations, and decanting ortopical applications during surgery. For example, direct injection ofnaked calcium phosphate-precipitated plasmid into rat liver and ratspleen or a protein-coated plasmid into the portal vein has resulted ingene expression of the foreign gene in the rat livers. (Kaneda et al.,Science, 243:375 (1989)).

A preferred method of local administration is by direct injection.Preferably, a recombinant molecule of the present invention complexedwith a delivery vehicle is administered by direct injection into orlocally within the area of arteries. Administration of a compositionlocally within the area of arteries refers to injecting the compositioncentimeters and preferably, millimeters within arteries.

Another method of local administration is to contact a polynucleotideconstruct of the present invention in or around a surgical wound. Forexample, a patient can undergo surgery and the polynucleotide constructcan be coated on the surface of tissue inside the wound or the constructcan be injected into areas of tissue inside the wound.

Therapeutic compositions useful in systemic administration, includerecombinant molecules of the present invention complexed to a targeteddelivery vehicle of the present invention. Suitable delivery vehiclesfor use with systemic administration comprise liposomes comprisingligands for targeting the vehicle to a particular site.

Preferred methods of systemic administration, include intravenousinjection, aerosol, oral and percutaneous (topical) delivery.Intravenous injections can be performed using methods standard in theart. Aerosol delivery can also be performed using methods standard inthe art (see, for example, Stribling et al., Proc. Natl. Acad. Sci. USA,189:11277-11281 (1992), which is incorporated herein by reference). Oraldelivery can be performed by complexing a polynucleotide construct ofthe present invention to a carrier capable of withstanding degradationby digestive enzymes in the gut of an animal. Examples of such carriers,include plastic capsules or tablets, such as those known in the art.Topical delivery can be performed by mixing a polynucleotide constructof the present invention with a lipophilic reagent (e.g., DMSO) that iscapable of passing into the skin.

Determining an effective amount of substance to be delivered can dependupon a number of factors including, for example, the chemical structureand biological activity of the substance, the age and weight of theanimal, the precise condition requiring treatment and its severity, andthe route of administration. The frequency of treatments depends upon anumber of factors, such as the amount of polynucleotide constructsadministered per dose, as well as the health and history of the subject.The precise amount, number of doses, and timing of doses will bedetermined by the attending physician or veterinarian. Therapeuticcompositions of the present invention can be administered to any animal,preferably to mammals and birds. Preferred mammals include humans, dogs,cats, mice, rats, rabbits sheep, cattle, horses and pigs, with humansbeing particularly preferred.

Biological Activities

The polynucleotides or polypeptides, or agonists or antagonists of thepresent invention can be used in assays to test for one or morebiological activities. If these polynucleotides and polypeptides doexhibit activity in a particular assay, it is likely that thesemolecules may be involved in the diseases associated with the biologicalactivity. Thus, the polynucleotides or polypeptides, or agonists orantagonists could be used to treat the associated disease.

Immune Activity

The polynucleotides or polypeptides, or agonists or antagonists of thepresent invention may be useful in treating, preventing, and/ordiagnosing diseases, disorders, and/or conditions of the immune system,by activating or inhibiting the proliferation, differentiation, ormobilization (chemotaxis) of immune cells. Immune cells develop througha process called hematopoiesis, producing myeloid (platelets, red bloodcells, neutrophils, and macrophages) and lymphoid (B and T lymphocytes)cells from pluripotent stem cells. The etiology of these immunediseases, disorders, and/or conditions may be genetic, somatic, such ascancer or some autoimmune diseases, disorders, and/or conditions,acquired (e.g., by chemotherapy or toxins), or infectious. Moreover, apolynucleotides or polypeptides, or agonists or antagonists of thepresent invention can be used as a marker or detector of a particularimmune system disease or disorder.

A polynucleotides or polypeptides, or agonists or antagonists of thepresent invention may be useful in treating, preventing, and/ordiagnosing diseases, disorders, and/or conditions of hematopoieticcells. A polynucleotides or polypeptides, or agonists or antagonists ofthe present invention could be used to increase differentiation andproliferation of hematopoietic cells, including the pluripotent stemcells, in an effort to treat or prevent those diseases, disorders,and/or conditions associated with a decrease in certain (or many) typeshematopoietic cells. Examples of immunologic deficiency syndromesinclude, but are not limited to: blood protein diseases, disorders,and/or conditions (e.g. agammaglobulinemia, dysgammaglobulinemia),ataxia telangiectasia, common variable immunodeficiency, DigeorgeSyndrome, HIV infection, HTLV-BLV infection, leukocyte adhesiondeficiency syndrome, lymphopenia, phagocyte bactericidal dysfunction,severe combined immunodeficiency (SCIDs), Wiskott-Aldrich Disorder,anemia, thrombocytopenia, or hemoglobinuria.

Moreover, a polynucleotides or polypeptides, or agonists or antagonistsof the present invention could also be used to modulate hemostatic (thestopping of bleeding) or thrombolytic activity (clot formation). Forexample, by increasing hemostatic or thrombolytic activity, apolynucleotides or polypeptides, or agonists or antagonists of thepresent invention could be used to treat or prevent blood coagulationdiseases, disorders, and/or conditions (e.g., afibrinogenemia, factordeficiencies, arterial thrombosis, venous thrombosis, etc.), bloodplatelet diseases, disorders, and/or conditions (e.g. thrombocytopenia),or wounds resulting from trauma, surgery, or other causes.Alternatively, a polynucleotides or polypeptides, or agonists orantagonists of the present invention that can decrease hemostatic orthrombolytic activity could be used to inhibit or dissolve clotting.Polynucleotides or polypeptides, or agonists or antagonists of thepresent invention are may also be useful for the detection, prognosis,treatment, and/or prevention of heart attacks (infarction), strokes,scarring, fibrinolysis, uncontrolled bleeding, uncontrolled coagulation,uncontrolled complement fixation, and/or inflammation.

A polynucleotides or polypeptides, or agonists or antagonists of thepresent invention may also be useful in treating, preventing, and/ordiagnosing autoimmune diseases, disorders, and/or conditions. Manyautoimmune diseases, disorders, and/or conditions result frominappropriate recognition of self as foreign material by immune cells.This inappropriate recognition results in an immune response leading tothe destruction of the host tissue. Therefore, the administration of apolynucleotides or polypeptides, or agonists or antagonists of thepresent invention that inhibits an immune response, particularly theproliferation, differentiation, or chemotaxis of T-cells, may be aneffective therapy in preventing autoimmune diseases, disorders, and/orconditions.

Examples of autoimmune diseases, disorders, and/or conditions that canbe treated, prevented, and/or diagnosed or detected by the presentinvention include, but are not limited to: Addison's Disease, hemolyticanemia, antiphospholipid syndrome, rheumatoid arthritis, dermatitis,allergic encephalomyelitis, glomerulonephritis, Goodpasture's Syndrome,Graves' Disease, Multiple Sclerosis, Myasthenia Gravis, Neuritis,Ophthalmia, Bullous Pemphigoid, Pemphigus, Polyendocrinopathies,Purpura, Reiter's Disease, Stiff-Man Syndrome, Autoimmune Thyroiditis,Systemic Lupus Erythematosus, Autoimmune Pulmonary Inflammation,Guillain-Barre Syndrome, insulin dependent diabetes mellitis, andautoimmune inflammatory eye disease.

Similarly, allergic reactions and conditions, such as asthma(particularly allergic asthma) or other respiratory problems, may alsobe treated, prevented, and/or diagnosed by polynucleotides orpolypeptides, or agonists or antagonists of the present invention.Moreover, these molecules can be used to treat anaphylaxis,hypersensitivity to an antigenic molecule, or blood groupincompatibility.

A polynucleotides or polypeptides, or agonists or antagonists of thepresent invention may also be used to treat, prevent, and/or diagnoseorgan rejection or graft-versus-host disease (GVHD). Organ rejectionoccurs by host immune cell destruction of the transplanted tissuethrough an immune response. Similarly, an immune response is alsoinvolved in GVHD, but, in this case, the foreign transplanted immunecells destroy the host tissues. The administration of a polynucleotidesor polypeptides, or agonists or antagonists of the present inventionthat inhibits an immune response, particularly the proliferation,differentiation, or chemotaxis of T-cells, may be an effective therapyin preventing organ rejection or GVHD.

Similarly, a polynucleotides or polypeptides, or agonists or antagonistsof the present invention may also be used to modulate inflammation. Forexample, the polypeptide or polynucleotide or agonists or antagonist mayinhibit the proliferation and differentiation of cells involved in aninflammatory response. These molecules can be used to treat, prevent,and/or diagnose inflammatory conditions, both chronic and acuteconditions, including chronic prostatitis, granulomatous prostatitis andmalacoplakia, inflammation associated with infection (e.g., septicshock, sepsis, or systemic inflammatory response syndrome (SIRS)),ischemia-reperfusion injury, endotoxin lethality, arthritis,complement-mediated hyperacute rejection, nephritis, cytokine orchemokine induced lung injury, inflammatory bowel disease, Crohn'sdisease, or resulting from over production of cytokines (e.g., TNF orIL-1.)

Hyperproliferative Disorders

A polynucleotides or polypeptides, or agonists or antagonists of theinvention can be used to treat, prevent, and/or diagnosehyperproliferative diseases, disorders, and/or conditions, includingneoplasms. A polynucleotides or polypeptides, or agonists or antagonistsof the present invention may inhibit the proliferation of the disorderthrough direct or indirect interactions. Alternatively, apolynucleotides or polypeptides, or agonists or antagonists of thepresent invention may proliferate other cells which can inhibit thehyperproliferative disorder.

For example, by increasing an immune response, particularly increasingantigenic qualities of the hyperproliferative disorder or byproliferating, differentiating, or mobilizing T-cells,hyperproliferative diseases, disorders, and/or conditions can betreated, prevented, and/or diagnosed. This immune response may beincreased by either enhancing an existing immune response, or byinitiating a new immune response. Alternatively, decreasing an immuneresponse may also be a method of treating, preventing, and/or diagnosinghyperproliferative diseases, disorders, and/or conditions, such as achemotherapeutic agent.

Examples of hyperproliferative diseases, disorders, and/or conditionsthat can be treated, prevented, and/or diagnosed by polynucleotides orpolypeptides, or agonists or antagonists of the present inventioninclude, but are not limited to neoplasms located in the: colon,abdomen, bone, breast, digestive system, liver, pancreas, peritoneum,endocrine glands (adrenal, parathyroid, pituitary, testicles, ovary,thymus, thyroid), eye, head and neck, nervous (central and peripheral),lymphatic system, pelvic, skin, soft tissue, spleen, thoracic, andurogenital.

Similarly, other hyperproliferative diseases, disorders, and/orconditions can also be treated, prevented, and/or diagnosed by apolynucleotides or polypeptides, or agonists or antagonists of thepresent invention. Examples of such hyperproliferative diseases,disorders, and/or conditions include, but are not limited to:hypergammaglobulinemia, lymphoproliferative diseases, disorders, and/orconditions, paraproteinemias, purpura, sarcoidosis, Sezary Syndrome,Waldenstron's Macroglobulinemia, Gaucher's Disease, histiocytosis, andany other hyperproliferative disease, besides neoplasia, located in anorgan system listed above.

One preferred embodiment utilizes polynucleotides of the presentinvention to inhibit aberrant cellular division, by gene therapy usingthe present invention, and/or protein fusions or fragments thereof.

Thus, the present invention provides a method for treating or preventingcell proliferative diseases, disorders, and/or conditions by insertinginto an abnormally proliferating cell a polynucleotide of the presentinvention, wherein said polynucleotide represses said expression.

Another embodiment of the present invention provides a method oftreating or preventing cell-proliferative diseases, disorders, and/orconditions in individuals comprising administration of one or moreactive gene copies of the present invention to an abnormallyproliferating cell or cells. In a preferred embodiment, polynucleotidesof the present invention is a DNA construct comprising a recombinantexpression vector effective in expressing a DNA sequence encoding saidpolynucleotides. In another preferred embodiment of the presentinvention, the DNA construct encoding the polynucleotides of the presentinvention is inserted into cells to be treated utilizing a retrovirus,or more Preferably an adenoviral vector (See G J. Nabel, et. al., PNAS1999 96: 324-326, which is hereby incorporated by reference). In a mostpreferred embodiment, the viral vector is defective and will nottransform non-proliferating cells, only proliferating cells. Moreover,in a preferred embodiment, the polynucleotides of the present inventioninserted into proliferating cells either alone, or in combination withor fused to other polynucleotides, can then be modulated via an externalstimulus (i.e. magnetic, specific small molecule, chemical, or drugadministration, etc.), which acts upon the promoter upstream of saidpolynucleotides to induce expression of the encoded protein product. Assuch the beneficial therapeutic affect of the present invention may beexpressly modulated (i.e. to increase, decrease, or inhibit expressionof the present invention) based upon said external stimulus.

Polynucleotides of the present invention may be useful in repressingexpression of oncogenic genes or antigens. By “repressing expression ofthe oncogenic genes” is intended the suppression of the transcription ofthe gene, the degradation of the gene transcript (pre-message RNA), theinhibition of splicing, the destruction of the messenger RNA, theprevention of the post-translational modifications of the protein, thedestruction of the protein, or the inhibition of the normal function ofthe protein.

For local administration to abnormally proliferating cells,polynucleotides of the present invention may be administered by anymethod known to those of skill in the art including, but not limited totransfection, electroporation, microinjection of cells, or in vehiclessuch as liposomes, lipofectin, or as naked polynucleotides, or any othermethod described throughout the specification. The polynucleotide of thepresent invention may be delivered by known gene delivery systems suchas, but not limited to, retroviral vectors (Gilboa, J. Virology 44:845(1982); Hocke, Nature 320:275 (1986); Wilson, et al., Proc. Natl. Acad.Sci. U.S.A. 85:3014), vaccinia virus system (Chakrabarty et al., Mol.Cell Biol. 5:3403 (1985) or other efficient DNA delivery systems (Yateset al., Nature 313:812 (1985)) known to those skilled in the art. Thesereferences are exemplary only and are hereby incorporated by reference.In order to specifically deliver or transfect cells which are abnormallyproliferating and spare non-dividing cells, it is preferable to utilizea retrovirus, or adenoviral (as described in the art and elsewhereherein) delivery system known to those of skill in the art. Since hostDNA replication is required for retroviral DNA to integrate and theretrovirus will be unable to self replicate due to the lack of theretrovirus genes needed for its life cycle. Utilizing such a retroviraldelivery system for polynucleotides of the present invention will targetsaid gene and constructs to abnormally proliferating cells and willspare the non-dividing normal cells.

The polynucleotides of the present invention may be delivered directlyto cell proliferative disorder/disease sites in internal organs, bodycavities and the like by use of imaging devices used to guide aninjecting needle directly to the disease site. The polynucleotides ofthe present invention may also be administered to disease sites at thetime of surgical intervention.

By “cell proliferative disease” is meant any human or animal disease ordisorder, affecting any one or any combination of organs, cavities, orbody parts, which is characterized by single or multiple local abnormalproliferations of cells, groups of cells, or tissues, whether benign ormalignant.

Any amount of the polynucleotides of the present invention may beadministered as long as it has a biologically inhibiting effect on theproliferation of the treated cells. Moreover, it is possible toadminister more than one of the polynucleotide of the present inventionsimultaneously to the same site. By “biologically inhibiting” is meantpartial or total growth inhibition as well as decreases in the rate ofproliferation or growth of the cells. The biologically inhibitory dosemay be determined by assessing the effects of the polynucleotides of thepresent invention on target malignant or abnormally proliferating cellgrowth in tissue culture, tumor growth in animals and cell cultures, orany other method known to one of ordinary skill in the art.

The present invention is further directed to antibody-based therapieswhich involve administering of anti-polypeptides and anti-polynucleotideantibodies to a mammalian, preferably human, patient for treating,preventing, and/or diagnosing one or more of the described diseases,disorders, and/or conditions. Methods for producing anti-polypeptidesand anti-polynucleotide antibodies polyclonal and monoclonal antibodiesare described in detail elsewhere herein. Such antibodies may beprovided in pharmaceutically acceptable compositions as known in the artor as described herein.

A summary of the ways in which the antibodies of the present inventionmay be used therapeutically includes binding polynucleotides orpolypeptides of the present invention locally or systemically in thebody or by direct cytotoxicity of the antibody, e.g. as mediated bycomplement (CDC) or by effector cells (ADCC). Some of these approachesare described in more detail below. Armed with the teachings providedherein, one of ordinary skill in the art will know how to use theantibodies of the present invention for diagnostic, monitoring ortherapeutic purposes without undue experimentation.

In particular, the antibodies, fragments and derivatives of the presentinvention are useful for treating, preventing, and/or diagnosing asubject having or developing cell proliferative and/or differentiationdiseases, disorders, and/or conditions as described herein. Suchtreatment comprises administering a single or multiple doses of theantibody, or a fragment, derivative, or a conjugate thereof.

The antibodies of this invention may be advantageously utilized incombination with other monoclonal or chimeric antibodies, or withlymphokines or hematopoietic growth factors, for example, which serve toincrease the number or activity of effector cells which interact withthe antibodies.

It is preferred to use high affinity and/or potent in vivo inhibitingand/or neutralizing antibodies against polypeptides or polynucleotidesof the present invention, fragments or regions thereof, for bothimmunoassays directed to and therapy of diseases, disorders, and/orconditions related to polynucleotides or polypeptides, includingfragments thereof, of the present invention. Such antibodies, fragments,or regions, will preferably have an affinity for polynucleotides orpolypeptides, including fragments thereof. Preferred binding affinitiesinclude those with a dissociation constant or Kd less than 5×10-6M,10-6M, 5×10-7M, 10-7M, 5×10-8M, 10-8M, 5×10-9M, 10-9M, 5×10-10M, 10-10M,5×10-11M, 10-11M, 5×10-12M, 10-12M, 5×10-13M, 10-13M, 5×10-14M, 10-14M,5×10-15M, and 10-15M.

Moreover, polypeptides of the present invention may be useful ininhibiting the angiogenesis of proliferative cells or tissues, eitheralone, as a protein fusion, or in combination with other polypeptidesdirectly or indirectly, as described elsewhere herein. In a mostpreferred embodiment, said anti-angiogenesis effect may be achievedindirectly, for example, through the inhibition of hematopoietic,tumor-specific cells, such as tumor-associated macrophages (See Joseph IB, et al. J Natl Cancer Inst, 90(21):1648-53 (1998), which is herebyincorporated by reference). Antibodies directed to polypeptides orpolynucleotides of the present invention may also result in inhibitionof angiogenesis directly, or indirectly (See Witte L, et al., CancerMetastasis Rev. 17(2):155-61 (1998), which is hereby incorporated byreference)).

Polypeptides, including protein fusions, of the present invention, orfragments thereof may be useful in inhibiting proliferative cells ortissues through the induction of apoptosis. Said polypeptides may acteither directly, or indirectly to induce apoptosis of proliferativecells and tissues, for example in the activation of a death-domainreceptor, such as tumor necrosis factor (TNF) receptor-1, CD95(Fas/APO-1), TNF-receptor-related apoptosis-mediated protein (TRAMP) andTNF-related apoptosis-inducing ligand (TRAIL) receptor-1 and -2 (SeeSchulze-Osthoff K, et al., Eur J Biochem 254(3):439-59 (1998), which ishereby incorporated by reference). Moreover, in another preferredembodiment of the present invention, said polypeptides may induceapoptosis through other mechanisms, such as in the activation of otherproteins which will activate apoptosis, or through stimulating theexpression of said proteins, either alone or in combination with smallmolecule drugs or adjuvants, such as apoptonin, galectins, thioredoxins,antiinflammatory proteins (See for example, Mutat. Res. 400(1-2):447-55(1998), Med Hypotheses.50(5):423-33 (1998), Chem. Biol. Interact. April24;111-112:23-34 (1998), J Mol Med.76(6):402-12 (1998), Int. J. TissueReact. 20(1):3-15 (1998), which are all hereby incorporated byreference).

Polypeptides, including protein fusions to, or fragments thereof, of thepresent invention are useful in inhibiting the metastasis ofproliferative cells or tissues. Inhibition may occur as a direct resultof administering polypeptides, or antibodies directed to saidpolypeptides as described elsewhere herein, or indirectly, such asactivating the expression of proteins known to inhibit metastasis, forexample alpha 4 integrins, (See, e.g., Curr Top Microbiol Immunol1998;231:125-41, which is hereby incorporated by reference). Suchtherapeutic affects of the present invention may be achieved eitheralone, or in combination with small molecule drugs or adjuvants.

In another embodiment, the invention provides a method of deliveringcompositions containing the polypeptides of the invention (e.g.,compositions containing polypeptides or polypeptide antibodiesassociated with heterologous polypeptides, heterologous nucleic acids,toxins, or prodrugs) to targeted cells expressing the polypeptide of thepresent invention. Polypeptides or polypeptide antibodies of theinvention may be associated with heterologous polypeptides, heterologousnucleic acids, toxins, or prodrugs via hydrophobic, hydrophilic, ionicand/or covalent interactions.

Polypeptides, protein fusions to, or fragments thereof, of the presentinvention are useful in enhancing the immunogenicity and/or antigenicityof proliferating cells or tissues, either directly, such as would occurif the polypeptides of the present invention ‘vaccinated’ the immuneresponse to respond to proliferative antigens and immunogens, orindirectly, such as in activating the expression of proteins known toenhance the immune response (e.g. chemokines), to said antigens andimmunogens.

Cardiovascular Disorders

Polynucleotides or polypeptides, or agonists or antagonists of theinvention may be used to treat, prevent, and/or diagnose cardiovasculardiseases, disorders, and/or conditions, including peripheral arterydisease, such as limb ischemia.

Cardiovascular diseases, disorders, and/or conditions includecardiovascular abnormalities, such as arterio-arterial fistula,arteriovenous fistula, cerebral arteriovenous malformations, congenitalheart defects, pulmonary atresia, and Scimitar Syndrome. Congenitalheart defects include aortic coarctation, cor triatriatum, coronaryvessel anomalies, crisscross heart, dextrocardia, patent ductusarteriosus, Ebstein's anomaly, Eisenmenger complex, hypoplastic leftheart syndrome, levocardia, tetralogy of fallot, transposition of greatvessels, double outlet right ventricle, tricuspid atresia, persistenttruncus arteriosus, and heart septal defects, such as aortopulnonaryseptal defect, endocardial cushion defects, Lutembacher's Syndrome,trilogy of Fallot, ventricular heart septal defects.

Cardiovascular diseases, disorders, and/or conditions also include heartdisease, such as arrhythmias, carcinoid heart disease, high cardiacoutput, low cardiac output, cardiac tamponade, endocarditis (includingbacterial), heart aneurysm, cardiac arrest, congestive heart failure,congestive cardiomyopathy, paroxysmal dyspnea, cardiac edema, hearthypertrophy, congestive cardiomyopathy, left ventricular hypertrophy,right ventricular hypertrophy, post-infarction heart rupture,ventricular septal rupture, heart valve diseases, myocardial diseases,myocardial ischemia, pericardial effusion, pericarditis (includingconstrictive and tuberculous), pneumopericardium, postpericardiotomysyndrome, pulmonary heart disease, rheumatic heart disease, ventriculardysfunction, hyperemia, cardiovascular pregnancy complications, ScimitarSyndrome, cardiovascular syphilis, and cardiovascular tuberculosis.

Arrhythmias include sinus arrhythmia, atrial fibrillation, atrialflutter, bradycardia, extrasystole, Adams-Stokes Syndrome, bundle-branchblock, sinoatrial block, long QT syndrome, parasystole,Lown-Ganong-Levine Syndrome, Mahaim-type pre-excitation syndrome,Wolff-Parkinson-White syndrome, sick sinus syndrome, tachycardias, andventricular fibrillation. Tachycardias include paroxysmal tachycardia,supraventricular tachycardia, accelerated idioventricular rhythm,atrioventricular nodal reentry tachycardia, ectopic atrial tachycardia,ectopic junctional tachycardia, sinoatrial nodal reentry tachycardia,sinus tachycardia, Torsades de Pointes, and ventricular tachycardia.

Heart valve disease include aortic valve insufficiency, aortic valvestenosis, hear murmurs, aortic valve prolapse, mitral valve prolapse,tricuspid valve prolapse, mitral valve insufficiency, mitral valvestenosis, pulmonary atresia, pulmonary valve insufficiency, pulmonaryvalve stenosis, tricuspid atresia, tricuspid valve insufficiency, andtricuspid valve stenosis.

Myocardial diseases include alcoholic cardiomyopathy, congestivecardiomyopathy, hypertrophic cardiomyopathy, aortic subvalvularstenosis, pulmonary subvalvular stenosis, restrictive cardiomyopathy,Chagas cardiomyopathy, endocardial fibroelastosis, endomyocardialfibrosis, Kearns Syndrome, myocardial reperfusion injury, andmyocarditis.

Myocardial ischemias include coronary disease, such as angina pectoris,coronary aneurysm, coronary arteriosclerosis, coronary thrombosis,coronary vasospasm, myocardial infarction and myocardial stunning.

Cardiovascular diseases also include vascular diseases such asaneurysms, angiodysplasia, angiomatosis, bacillary angiomatosis,Hippel-Lindau Disease, Klippel-Trenaunay-Weber Syndrome, Sturge-WeberSyndrome, angioneurotic edema, aortic diseases, Takayasu's Arteritis,aortitis, Leriche's Syndrome, arterial occlusive diseases, arteritis,enarteritis, polyarteritis nodosa, cerebrovascular diseases, disorders,and/or conditions, diabetic angiopathies, diabetic retinopathy,embolisms, thrombosis, erythromelalgia, hemorrhoids, hepaticveno-occlusive disease, hypertension, hypotension, ischemia, peripheralvascular diseases, phlebitis, pulmonary veno-occlusive disease,Raynaud's disease, CREST syndrome, retinal vein occlusion, Scimitarsyndrome, superior vena cava syndrome, telangiectasia, ataciatelangiectasia, hereditary hemorrhagic telangiectasia, varicocele,varicose veins, varicose ulcer, vasculitis, and venous insufficiency.

Aneurysms include dissecting aneurysms, false aneurysms, infectedaneurysms, ruptured aneurysms, aortic aneurysms, cerebral aneurysms,coronary aneurysms, heart aneurysms, and iliac aneurysms.

Arterial occlusive diseases include arteriosclerosis, intermittentclaudication, carotid stenosis, fibromuscular dysplasias, mesentericvascular occlusion, Moyamoya disease, renal artery obstruction, retinalartery occlusion, and thromboangiitis obliterans.

Cerebrovascular diseases, disorders, and/or conditions include carotidartery diseases, cerebral amyloid angiopathy, cerebral aneurysm,cerebral anoxia, cerebral arteriosclerosis, cerebral arteriovenousmalformation, cerebral artery diseases, cerebral embolism andthrombosis, carotid artery thrombosis, sinus thrombosis, Wallenberg'ssyndrome, cerebral hemorrhage, epidural hematoma, subdural hematoma,subaraxhnoid hemorrhage, cerebral infarction, cerebral ischemia(including transient), subclavian steal syndrome, periventricularleukomalacia, vascular headache, cluster headache, migraine, andvertebrobasilar insufficiency.

Embolisms include air embolisms, amniotic fluid embolisms, cholesterolembolisms, blue toe syndrome, fat embolisms, pulmonary embolisms, andthromoboembolisms. Thrombosis include coronary thrombosis, hepatic veinthrombosis, retinal vein occlusion, carotid artery thrombosis, sinusthrombosis, Wallenberg's syndrome, and thrombophlebitis.

Ischemia includes cerebral ischemia, ischemic colitis, compartmentsyndromes, anterior compartment syndrome, myocardial ischemia,reperfusion injuries, and peripheral limb ischemia. Vasculitis includesaortitis, arteritis, Behcet's Syndrome, Churg-Strauss Syndrome,mucocutaneous lymph node syndrome, thromboangiitis obliterans,hypersensitivity vasculitis, Schoenlein-Henoch purpura, allergiccutaneous vasculitis, and Wegener's granulomatosis.

Polynucleotides or polypeptides, or agonists or antagonists of theinvention, are especially effective for the treatment of critical limbischemia and coronary disease.

Polypeptides may be administered using any method known in the art,including, but not limited to, direct needle injection at the deliverysite, intravenous injection, topical administration, catheter infusion,biolistic injectors, particle accelerators, gelfoam sponge depots, othercommercially available depot materials, osmotic pumps, oral orsuppositorial solid pharmaceutical formulations, decanting or topicalapplications during surgery, aerosol delivery. Such methods are known inthe art. Polypeptides of the invention may be administered as part of aTherapeutic, described in more detail below. Methods of deliveringpolynucleotides of the invention are described in more detail herein.

Diseases at the Cellular Level

Diseases associated with increased cell survival or the inhibition ofapoptosis that could be treated, prevented, and/or diagnosed by thepolynucleotides or polypeptides and/or antagonists or agonists of theinvention, include cancers (such as follicular lymphomas, carcinomaswith p53 mutations, and hormone-dependent tumors, including, but notlimited to colon cancer, cardiac tumors, pancreatic cancer, melanoma,retinoblastoma, glioblastoma, lung cancer, intestinal cancer, testicularcancer, stomach cancer, neuroblastoma, myxoma, myoma, lymphoma,endothelioma, osteoblastoma, osteoclastoma, osteosarcoma,chondrosarcoma, adenoma, breast cancer, prostate cancer, Kaposi'ssarcoma and ovarian cancer); autoimmune diseases, disorders, and/orconditions (such as, multiple sclerosis, Sjogren's syndrome, Hashimoto'sthyroiditis, biliary cirrhosis, Behcet's disease, Crohn's disease,polymyositis, systemic lupus erythematosus and immune-relatedglomerulonephritis and rheumatoid arthritis) and viral infections (suchas herpes viruses, pox viruses and adenoviruses), inflammation, graft v.host disease, acute graft rejection, and chronic graft rejection. Inpreferred embodiments, the polynucleotides or polypeptides, and/oragonists or antagonists of the invention are used to inhibit growth,progression, and/or metastasis of cancers, in particular those listedabove.

Additional diseases or conditions associated with increased cellsurvival that could be treated, prevented or diagnosed by thepolynucleotides or polypeptides, or agonists or antagonists of theinvention, include, but are not limited to, progression, and/ormetastases of malignancies and related disorders such as leukemia(including acute leukemias (e.g., acute lymphocytic leukemia, acutemyelocytic leukemia (including myeloblastic, promyelocytic,myelomonocytic, monocytic, and erythroleukemia)) and chronic leukemias(e.g., chronic myelocytic (granulocytic) leukemia and chroniclymphocytic leukemia)), polycythemia vera, lymphomas (e.g., Hodgkin'sdisease and non-Hodgkin's disease), multiple myeloma, Waldenstrom'smacroglobulinemia, heavy chain disease, and solid tumors including, butnot limited to, sarcomas and carcinomas such as fibrosarcoma,myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma,angiosarcoma, endotheliosarcoma, lymphangiosarcoma,lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor,leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer,breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma,basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceousgland carcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, testiculartumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma,epithelial carcinoma, glioma, astrocytoma, medulloblastoma,craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acousticneuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma, andretinoblastoma.

Diseases associated with increased apoptosis that could be treated,prevented, and/or diagnosed by the polynucleotides or polypeptides,and/or agonists or antagonists of the invention, include AIDS;neurodegenerative diseases, disorders, and/or conditions (such asAlzheimer's disease, Parkinson's disease, Amyotrophic lateral sclerosis,Retinitis pigmentosa, Cerebellar degeneration and brain tumor or priorassociated disease); autoimmune diseases, disorders, and/or conditions(such as, multiple sclerosis, Sjogren's syndrome, Hashimoto'sthyroiditis, biliary cirrhosis, Behcet's disease, Crohn's disease,polymyositis, systemic lupus erythematosus and immune-relatedglomerulonephritis and rheumatoid arthritis) myelodysplastic syndromes(such as aplastic anemia), graft v. host disease, ischemic injury (suchas that caused by myocardial infarction, stroke and reperfusion injury),liver injury (e.g., hepatitis related liver injury, ischemia/reperfusioninjury, cholestosis (bile duct injury) and liver cancer); toxin-inducedliver disease (such as that caused by alcohol), septic shock, cachexiaand anorexia.

Wound Healing and Epithelial Cell Proliferation

In accordance with yet a further aspect of the present invention, thereis provided a process for utilizing the polynucleotides or polypeptides,and/or agonists or antagonists of the invention, for therapeuticpurposes, for example, to stimulate epithelial cell proliferation andbasal keratinocytes for the purpose of wound healing, and to stimulatehair follicle production and healing of dermal wounds. Polynucleotidesor polypeptides, as well as agonists or antagonists of the invention,may be clinically useful in stimulating wound healing including surgicalwounds, excisional wounds, deep wounds involving damage of the dermisand epidermis, eye tissue wounds, dental tissue wounds, oral cavitywounds, diabetic ulcers, dermal ulcers, cubitus ulcers, arterial ulcers,venous stasis ulcers, burns resulting from heat exposure or chemicals,and other abnormal wound healing conditions such as uremia,malnutrition, vitamin deficiencies and complications associated withsystemic treatment with steroids, radiation therapy and antineoplasticdrugs and antimetabolites. Polynucleotides or polypeptides, and/oragonists or antagonists of the invention, could be used to promotedermal reestablishment subsequent to dermal loss The polynucleotides orpolypeptides, and/or agonists or antagonists of the invention, could beused to increase the adherence of skin grafts to a wound bed and tostimulate re-epithelialization from the wound bed. The following are anon-exhaustive list of grafts that polynucleotides or polypeptides,agonists or antagonists of the invention, could be used to increaseadherence to a wound bed: autografts, artificial skin, allografts,autodermic graft, autoepidermic grafts, avacular grafts, Blair-Browngrafts, bone graft, brephoplastic grafts, cutis graft, delayed graft,dermic graft, epidermic graft, fascia graft, full thickness graft,heterologous graft, xenograft, homologous graft, hyperplastic graft,lamellar graft, mesh graft, mucosal graft, Ollier-Thiersch graft,omenpal graft, patch graft, pedicle graft, penetrating graft, split skingraft, thick split graft. The polynucleotides or polypeptides, and/oragonists or antagonists of the invention, can be used to promote skinstrength and to improve the appearance of aged skin.

It is believed that the polynucleotides or polypeptides, and/or agonistsor antagonists of the invention, will also produce changes in hepatocyteproliferation, and epithelial cell proliferation in the lung, breast,pancreas, stomach, small intestine, and large intestine. Thepolynucleotides or polypeptides, and/or agonists or antagonists of theinvention, could promote proliferation of epithelial cells such assebocytes, hair follicles, hepatocytes, type II pneumocytes,mucin-producing goblet cells, and other epithelial cells and theirprogenitors contained within the skin, lung, liver, and gastrointestinaltract. The polynucleotides or polypeptides, and/or agonists orantagonists of the invention, may promote proliferation of endothelialcells, keratinocytes, and basal keratinocytes.

The polynucleotides or polypeptides, and/or agonists or antagonists ofthe invention, could also be used to reduce the side effects of guttoxicity that result from radiation, chemotherapy treatments or viralinfections. The polynucleotides or polypeptides, and/or agonists orantagonists of the invention, may have a cytoprotective effect on thesmall intestine mucosa. The polynucleotides or polypeptides, and/oragonists or antagonists of the invention, may also stimulate healing ofmucositis (mouth ulcers) that result from chemotherapy and viralinfections.

The polynucleotides or polypeptides, and/or agonists or antagonists ofthe invention, could further be used in full regeneration of skin infull and partial thickness skin defects, including burns, (i.e.,repopulation of hair follicles, sweat glands, and sebaceous glands),treatment of other skin defects such as psoriasis. The polynucleotidesor polypeptides, and/or agonists or antagonists of the invention, couldbe used to treat epidermolysis bullosa, a defect in adherence of theepidermis to the underlying dermis which results in frequent, open andpainful blisters by accelerating reepithelialization of these lesions.The polynucleotides or polypeptides, and/or agonists or antagonists ofthe invention, could also be used to treat gastric and doudenal ulcersand help heal by scar formation of the mucosal lining and regenerationof glandular mucosa and duodenal mucosal lining more rapidly.Inflamamatory bowel diseases, such as Crohn's disease and ulcerativecolitis, are diseases which result in destruction of the mucosal surfaceof the small or large intestine, respectively. Thus, the polynucleotidesor polypeptides, and/or agonists or antagonists of the invention, couldbe used to promote the resurfacing of the mucosal surface to aid morerapid healing and to prevent progression of inflammatory bowel disease.Treatment with the polynucleotides or polypeptides, and/or agonists orantagonists of the invention, is expected to have a significant effecton the production of mucus throughout the gastrointestinal tract andcould be used to protect the intestinal mucosa from injurious substancesthat are ingested or following surgery. The polynucleotides orpolypeptides, and/or agonists or antagonists of the invention, could beused to treat diseases associate with the under expression of thepolynucleotides of the invention.

Moreover, the polynucleotides or polypeptides, and/or agonists orantagonists of the invention, could be used to prevent and heal damageto the lungs due to various pathological states. A growth factor such asthe polynucleotides or polypeptides, and/or agonists or antagonists ofthe invention, which could stimulate proliferation and differentiationand promote the repair of alveoli and brochiolar epithelium to preventor treat acute or chronic lung damage. For example, emphysema, whichresults in the progressive loss of aveoli, and inhalation injuries,i.e., resulting from smoke inhalation and burns, that cause necrosis ofthe bronchiolar epithelium and alveoli could be effectively treated,prevented, and/or diagnosed using the polynucleotides or polypeptides,and/or agonists or antagonists of the invention. Also, thepolynucleotides or polypeptides, and/or agonists or antagonists of theinvention, could be used to stimulate the proliferation of anddifferentiation of type II pneumocytes, which may help treat or preventdisease such as hyaline membrane diseases, such as infant respiratorydistress syndrome and bronchopulmonary displasia, in premature infants.

The polynucleotides or polypeptides, and/or agonists or antagonists ofthe invention, could stimulate the proliferation and differentiation ofhepatocytes and, thus, could be used to alleviate or treat liverdiseases and pathologies such as fulminant liver failure caused bycirrhosis, liver damage caused by viral hepatitis and toxic substances(i.e., acetaminophen, carbon tetraholoride and other hepatotoxins knownin the art).

In addition, the polynucleotides or polypeptides, and/or agonists orantagonists of the invention, could be used treat or prevent the onsetof diabetes mellitus. In patients with newly diagnosed Types I and IIdiabetes, where some islet cell function remains, the polynucleotides orpolypeptides, and/or agonists or antagonists of the invention, could beused to maintain the islet function so as to alleviate, delay or preventpermanent manifestation of the disease. Also, the polynucleotides orpolypeptides, and/or agonists or antagonists of the invention, could beused as an auxiliary in islet cell transplantation to improve or promoteislet cell function.

Neurological Diseases

Nervous system diseases, disorders, and/or conditions, which can betreated, prevented, and/or diagnosed with the compositions of theinvention (e.g., polypeptides, polynucleotides, and/or agonists orantagonists), include, but are not limited to, nervous system injuries,and diseases, disorders, and/or conditions which result in either adisconnection of axons, a diminution or degeneration of neurons, ordemyelination. Nervous system lesions which may be treated, prevented,and/or diagnosed in a patient (including human and non-human mammalianpatients) according to the invention, include but are not limited to,the following lesions of either the central (including spinal cord,brain) or peripheral nervous systems: (1) ischemic lesions, in which alack of oxygen in a portion of the nervous system results in neuronalinjury or death, including cerebral infarction or ischemia, or spinalcord infarction or ischemia; (2) traumatic lesions, including lesionscaused by physical injury or associated with surgery, for example,lesions which sever a portion of the nervous system, or compressioninjuries; (3) malignant lesions, in which a portion of the nervoussystem is destroyed or injured by malignant tissue which is either anervous system associated malignancy or a malignancy derived fromnon-nervous system tissue; (4) infectious lesions, in which a portion ofthe nervous system is destroyed or injured as a result of infection, forexample, by an abscess or associated with infection by humanimmunodeficiency virus, herpes zoster, or herpes simplex virus or withLyme disease, tuberculosis, syphilis; (5) degenerative lesions, in whicha portion of the nervous system is destroyed or injured as a result of adegenerative process including but not limited to degenerationassociated with Parkinson's disease, Alzheimer's disease, Huntington'schorea, or amyotrophic lateral sclerosis (ALS); (6) lesions associatedwith nutritional diseases, disorders, and/or conditions, in which aportion of the nervous system is destroyed or injured by a nutritionaldisorder or disorder of metabolism including but not limited to, vitaminB 12 deficiency, folic acid deficiency, Wemicke disease, tobacco-alcoholamblyopia, Marchiafava-Bignami disease (primary degeneration of thecorpus callosum), and alcoholic cerebellar degeneration; (7)neurological lesions associated with systemic diseases including, butnot limited to, diabetes (diabetic neuropathy, Bell's palsy), systemiclupus erythematosus, carcinoma, or sarcoidosis; (8) lesions caused bytoxic substances including alcohol, lead, or particular neurotoxins; and(9) demyelinated lesions in which a portion of the nervous system isdestroyed or injured by a demyelinating disease including, but notlimited to, multiple sclerosis, human immunodeficiency virus-associatedmyelopathy, transverse myelopathy or various etiologies, progressivemultifocal leukoencephalopathy, and central pontine myelinolysis.

In a preferred embodiment, the polypeptides, polynucleotides, oragonists or antagonists of the invention are used to protect neuralcells from the damaging effects of cerebral hypoxia. According to thisembodiment, the compositions of the invention are used to treat,prevent, and/or diagnose neural cell injury associated with cerebralhypoxia. In one aspect of this embodiment, the polypeptides,polynucleotides, or agonists or antagonists of the invention are used totreat, prevent, and/or diagnose neural cell injury associated withcerebral ischemia. In another aspect of this embodiment, thepolypeptides, polynucleotides, or agonists or antagonists of theinvention are used to treat, prevent, and/or diagnose neural cell injuryassociated with cerebral infarction. In another aspect of thisembodiment, the polypeptides, polynucleotides, or agonists orantagonists of the invention are used to treat, prevent, and/or diagnoseor prevent neural cell injury associated with a stroke. In a furtheraspect of this embodiment, the polypeptides, polynucleotides, oragonists or antagonists of the invention are used to treat, prevent,and/or diagnose neural cell injury associated with a heart attack.

The compositions of the invention which are useful for treating orpreventing a nervous system disorder may be selected by testing forbiological activity in promoting the survival or differentiation ofneurons. For example, and not by way of limitation, compositions of theinvention which elicit any of the following effects may be usefulaccording to the invention: (1) increased survival time of neurons inculture; (2) increased sprouting of neurons in culture or in vivo; (3)increased production of a neuron-associated molecule in culture or invivo, e.g., choline acetyltransferase or acetylcholinesterase withrespect to motor neurons; or (4) decreased symptoms of neurondysfunction in vivo. Such effects may be measured by any method known inthe art. In preferred, non-limiting embodiments, increased survival ofneurons may routinely be measured using a method set forth herein orotherwise known in the art, such as, for example, the method set forthin Arakawa et al. (J. Neurosci. 10:3507-3515 (1990)); increasedsprouting of neurons may be detected by methods known in the art, suchas, for example, the methods set forth in Pestronk et al. (Exp. Neurol.70:65-82 (1980)) or Brown et al. (Ann. Rev. Neurosci. 4:17-42 (1981));increased production of neuron-associated molecules may be measured bybioassay, enzymatic assay, antibody binding, Northern blot assay, etc.,using techniques known in the art and depending on the molecule to bemeasured; and motor neuron dysfunction may be measured by assessing thephysical manifestation of motor neuron disorder, e.g., weakness, motorneuron conduction velocity, or functional disability.

In specific embodiments, motor neuron diseases, disorders, and/orconditions that may be treated, prevented, and/or diagnosed according tothe invention include, but are not limited to, diseases, disorders,and/or conditions such as infarction, infection, exposure to toxin,trauma, surgical damage, degenerative disease or malignancy that mayaffect motor neurons as well as other components of the nervous system,as well as diseases, disorders, and/or conditions that selectivelyaffect neurons such as amyotrophic lateral sclerosis, and including, butnot limited to, progressive spinal muscular atrophy, progressive bulbarpalsy, primary lateral sclerosis, infantile and juvenile muscularatrophy, progressive bulbar paralysis of childhood (Fazio-Londesyndrome), poliomyelitis and the post polio syndrome, and HereditaryMotorsensory Neuropathy (Charcot-Marie-Tooth Disease).

Infectious Disease

A polypeptide or polynucleotide and/or agonist or antagonist of thepresent invention can be used to treat, prevent, and/or diagnoseinfectious agents. For example, by increasing the immune response,particularly increasing the proliferation and differentiation of Band/or T cells, infectious diseases may be treated, prevented, and/ordiagnosed. The immune response may be increased by either enhancing anexisting immune response, or by initiating a new immune response.Alternatively, polypeptide or polynucleotide and/or agonist orantagonist of the present invention may also directly inhibit theinfectious agent, without necessarily eliciting an immune response.

Viruses are one example of an infectious agent that can cause disease orsymptoms that can be treated, prevented, and/or diagnosed by apolynucleotide or polypeptide and/or agonist or antagonist of thepresent invention. Examples of viruses, include, but are not limited toExamples of viruses, include, but are not limited to the following DNAand RNA viruses and viral families: Arbovirus, Adenoviridae,Arenaviridae, Arterivirus, Bimaviridae, Bunyaviridae, Caliciviridae,Circoviridae, Coronaviridae, Dengue, EBV, HIV, Flaviviridae,Hepadnaviridae (Hepatitis), Herpesviridae (such as, Cytomegalovirus,Herpes Simplex, Herpes Zoster), Mononegavirus (e.g., Paramyxoviridae,Morbillivirus, Rhabdoviridae), Orthomyxoviridae (e.g., Influenza A,Influenza B, and parainfluenza), Papiloma virus, Papovaviridae,Parvoviridae, Picornaviridae, Poxviridae (such as Smallpox or Vaccinia),Reoviridae (e.g., Rotavirus), Retroviridae (HTLV-I, HTLV-II,Lentivirus), and Togaviridae (e.g., Rubivirus). Viruses falling withinthese families can cause a variety of diseases or symptoms, including,but not limited to: arthritis, bronchiollitis, respiratory syncytialvirus, encephalitis, eye infections (e.g., conjunctivitis, keratitis),chronic fatigue syndrome, hepatitis (A, B, C, E, Chronic Active, Delta),Japanese B encephalitis, Junin, Chikungunya, Rift Valley fever, yellowfever, meningitis, opportunistic infections (e.g., AIDS), pneumonia,Burkitt's Lymphoma, chickenpox, hemorrhagic fever, Measles, Mumps,Parainfluenza, Rabies, the common cold, Polio, leukemia, Rubella,sexually transmitted diseases, skin diseases (e.g., Kaposi's, warts),and viremia polynucleotides or polypeptides, or agonists or antagonistsof the invention, can be used to treat, prevent, and/or diagnose any ofthese symptoms or diseases. In specific embodiments, polynucleotides,polypeptides, or agonists or antagonists of the invention are used totreat, prevent, and/or diagnose: meningitis, Dengue, EBV, and/orhepatitis (e.g., hepatitis B). In an additional specific embodimentpolynucleotides, polypeptides, or agonists or antagonists of theinvention are used to treat patients nonresponsive to one or more othercommercially available hepatitis vaccines. In a further specificembodiment polynucleotides, polypeptides, or agonists or antagonists ofthe invention are used to treat, prevent, and/or diagnose AIDS.

Similarly, bacterial or fungal agents that can cause disease or symptomsand that can be treated, prevented, and/or diagnosed by a polynucleotideor polypeptide and/or agonist or antagonist of the present inventioninclude, but not limited to, include, but not limited to, the followingGram-Negative and Gram-positive bacteria and bacterial families andfungi: Actinomycetales (e.g., Corynebacterium, Mycobacterium,Norcardia), Cryptococcus neoformans, Aspergillosis, Bacillaceae (e.g.,Anthrax, Clostridium), Bacteroidaceae, Blastomycosis, Bordetella,Borrelia (e.g., Borrelia burgdorferi), Brucellosis, Candidiasis,Campylobacter, Coccidioidomycosis, Cryptococcosis, Dermatocycoses, E.coli (e.g., Enterotoxigenic E. coli and Enterohemorrhagic E. coli),Enterobacteriaceae (Klebsiella, Salmonella (e.g., Salmonella typhi, andSalmonella paratyphi), Serratia, Yersinia), Erysipelothrix,Helicobacter, Legionellosis, Leptospirosis, Listeria, Mycoplasmatales,Mycobacterium leprae, Vibrio cholerae, Neisseriaceae (e.g.,Acinetobacter, Gonorrhea, Menigococcal), Meisseria meningitidis,Pasteurellacea Infections (e.g., Actinobacillus, Heamophilus (e.g.,Heamophilus influenza type B), Pasteurella), Pseudomonas,Rickettsiaceae, Chlamydiaceae, Syphilis, Shigella spp., Staphylococcal,Meningiococcal, Pneumococcal and Streptococcal (e.g., Streptococcuspneumoniae and Group B Streptococcus). These bacterial or fingalfamilies can cause the following diseases or symptoms, including, butnot limited to: bacteremia, endocarditis, eye infections(conjunctivitis, tuberculosis, uveitis), gingivitis, opportunisticinfections (e.g., AIDS related infections), paronychia,prosthesis-related infections, Reiter's Disease, respiratory tractinfections, such as Whooping Cough or Empyema, sepsis, Lyme Disease,Cat-Scratch Disease, Dysentery, Paratyphoid Fever, food poisoning,Typhoid, pneumonia, Gonorrhea, meningitis (e.g., mengitis types A andB), Chlamydia, Syphilis, Diphtheria, Leprosy, Paratuberculosis,Tuberculosis, Lupus, Botulism, gangrene, tetanus, impetigo, RheumaticFever, Scarlet Fever, sexually transmitted diseases, skin diseases(e.g., cellulitis, dermatocycoses), toxemia, urinary tract infections,wound infections. Polynucleotides or polypeptides, agonists orantagonists of the invention, can be used to treat, prevent, and/ordiagnose any of these symptoms or diseases. In specific embodiments,polynucleotides, polypeptides, agonists or antagonists of the inventionare used to treat, prevent, and/or diagnose: tetanus, Diptheria,botulism, and/or meningitis type B.

Moreover, parasitic agents causing disease or symptoms that can betreated, prevented, and/or diagnosed by a polynucleotide or polypeptideand/or agonist or antagonist of the present invention include, but notlimited to, the following families or class: Amebiasis, Babesiosis,Coccidiosis, Cryptosporidiosis, Dientamoebiasis, Dourine, Ectoparasitic,Giardiasis, Helminthiasis, Leishmaniasis, Theileriasis, Toxoplasmosis,Trypanosomiasis, and Trichomonas and Sporozoans (e.g., Plasmodium virax,Plasmodium falciparium, Plasmodium malariae and Plasmodium ovale). Theseparasites can cause a variety of diseases or symptoms, including, butnot limited to: Scabies, Trombiculiasis, eye infections, intestinaldisease (e.g., dysentery, giardiasis), liver disease, lung disease,opportunistic infections (e.g., AIDS related), malaria, pregnancycomplications, and toxoplasmosis. Polynucleotides or polypeptides, oragonists or antagonists of the invention, can be used totreat, prevent,and/or diagnose any of these symptoms or diseases. In specificembodiments, polynucleotides, polypeptides, or agonists or antagonistsof the invention are used to treat, prevent, and/or diagnose malaria.

Preferably, treatment or prevention using a polypeptide orpolynucleotide and/or agonist or antagonist of the present inventioncould either be by administering an effective amount of a polypeptide tothe patient, or by removing cells from the patient, supplying the cellswith a polynucleotide of the present invention, and returning theengineered cells to the patient (ex vivo therapy). Moreover, thepolypeptide or polynucleotide of the present invention can be used as anantigen in a vaccine to raise an immune response against infectiousdisease.

Regeneration

A polynucleotide or polypeptide and/or agonist or antagonist of thepresent invention can be used to differentiate, proliferate, and attractcells, leading to the regeneration of tissues. (See, Science 276:59-87(1997).) The regeneration of tissues could be used to repair, replace,or protect tissue damaged by congenital defects, trauma (wounds, burns,incisions, or ulcers), age, disease (e.g. osteoporosis, osteocarthritis,periodontal disease, liver failure), surgery, including cosmetic plasticsurgery, fibrosis, reperfusion injury, or systemic cytokine damage.

Tissues that could be regenerated using the present invention includeorgans (e.g., pancreas, liver, intestine, kidney, skin, endothelium),muscle (smooth, skeletal or cardiac), vasculature (including vascularand lymphatics), nervous, hematopoietic, and skeletal (bone, cartilage,tendon, and ligament) tissue. Preferably, regeneration occurs without ordecreased scarring. Regeneration also may include angiogenesis.

Moreover, a polynucleotide or polypeptide and/or agonist or antagonistof the present invention may increase regeneration of tissues difficultto heal. For example, increased tendon/ligament regeneration wouldquicken recovery time after damage. A polynucleotide or polypeptideand/or agonist or antagonist of the present invention could also be usedprophylactically in an effort to avoid damage. Specific diseases thatcould be treated, prevented, and/or diagnosed include of tendinitis,carpal tunnel syndrome, and other tendon or ligament defects. A furtherexample of tissue regeneration of non-healing wounds includes pressureulcers, ulcers associated with vascular insufficiency, surgical, andtraumatic wounds.

Similarly, nerve and brain tissue could also be regenerated by using apolynucleotide or polypeptide and/or agonist or antagonist of thepresent invention to proliferate and differentiate nerve cells. Diseasesthat could be treated, prevented, and/or diagnosed using this methodinclude central and peripheral nervous system diseases, neuropathies, ormechanical and traumatic diseases, disorders, and/or conditions (e.g.,spinal cord disorders, head trauma, cerebrovascular disease, and stoke).Specifically, diseases associated with peripheral nerve injuries,peripheral neuropathy (e.g., resulting from chemotherapy or othermedical therapies), localized neuropathies, and central nervous systemdiseases (e.g., Alzheimer's disease, Parkinson's disease, Huntington'sdisease, amyotrophic lateral sclerosis, and Shy-Drager syndrome), couldall be treated, prevented, and/or diagnosed using the polynucleotide orpolypeptide and/or agonist or antagonist of the present invention.

Binding Activity

A polypeptide of the present invention may be used to screen formolecules that bind to the polypeptide or for molecules to which thepolypeptide binds. The binding of the polypeptide and the molecule mayactivate (agonist), increase, inhibit (antagonist), or decrease activityof the polypeptide or the molecule bound. Examples of such moleculesinclude antibodies, oligonucleotides, proteins (e.g., receptors), orsmall molecules.

Preferably, the molecule is closely related to the natural ligand of thepolypeptide, e.g., a fragment of the ligand, or a natural substrate, aligand, a structural or functional mimetic. (See, Coligan et al.,Current Protocols in Immunology 1(2):Chapter 5 (1991).) Similarly, themolecule can be closely related to the natural receptor to which thepolypeptide binds, or at least, a fragment of the receptor capable ofbeing bound by the polypeptide (e.g., active site). In either case, themolecule can be rationally designed using known techniques.

Preferably, the screening for these molecules involves producingappropriate cells which express the polypeptide, either as a secretedprotein or on the cell membrane. Preferred cells include cells frommammals, yeast, Drosophila, or E. coli. Cells expressing the polypeptide(or cell membrane containing the expressed polypeptide) are thenpreferably contacted with a test compound potentially containing themolecule to observe binding, stimulation, or inhibition of activity ofeither the polypeptide or the molecule.

The assay may simply test binding of a candidate compound to thepolypeptide, wherein binding is detected by a label, or in an assayinvolving competition with a labeled competitor. Further, the assay maytest whether the candidate compound results in a signal generated bybinding to the polypeptide.

Alternatively, the assay can be carried out using cell-freepreparations, polypeptide/molecule affixed to a solid support, chemicallibraries, or natural product mixtures. The assay may also simplycomprise the steps of mixing a candidate compound with a solutioncontaining a polypeptide, measuring polypeptide/molecule activity orbinding, and comparing the polypeptide/molecule activity or binding to astandard.

Preferably, an ELISA assay can measure polypeptide level or activity ina sample (e.g., biological sample) using a monoclonal or polyclonalantibody. The antibody can measure polypeptide level or activity byeither binding, directly or indirectly, to the polypeptide or bycompeting with the polypeptide for a substrate.

Additionally, the receptor to which a polypeptide of the invention bindscan be identified by numerous methods known to those of skill in theart, for example, ligand panning and FACS sorting (Coligan, et al.,Current Protocols in Immun., 1(2), Chapter 5, (1991)). For example,expression cloning is employed wherein polyadenylated RNA is preparedfrom a cell responsive to the polypeptides, for example, NIH3T3 cellswhich are known to contain multiple receptors for the FGF familyproteins, and SC-3 cells, and a cDNA library created from this RNA isdivided into pools and used to transfect COS cells or other cells thatare not responsive to the polypeptides. Transfected cells which aregrown on glass slides are exposed to the polypeptide of the presentinvention, after they have been labeled. The polypeptides can be labeledby a variety of means including iodination or inclusion of a recognitionsite for a site-specific protein kinase.

Following fixation and incubation, the slides are subjected toauto-radiographic analysis. Positive pools are identified and sub-poolsare prepared and re-transfected using an iterative sub-pooling andre-screening process, eventually yielding a single clones that encodesthe putative receptor.

As an alternative approach for receptor identification, the labeledpolypeptides can be photoaffinity linked with cell membrane or extractpreparations that express the receptor molecule. Cross-linked materialis resolved by PAGE analysis and exposed to X-ray film. The labeledcomplex containing the receptors of the polypeptides can be excised,resolved into peptide fragments, and subjected to proteinmicrosequencing. The amino acid sequence obtained from microsequencingwould be used to design a set of degenerate oligonucleotide probes toscreen a cDNA library to identify the genes encoding the putativereceptors.

Moreover, the techniques of gene-shuffling, motif-shuffling,exon-shuffling, and/or codon-shuffling (collectively referred to as “DNAshuffling”) may be employed to modulate the activities of polypeptidesof the invention thereby effectively generating agonists and antagonistsof polypeptides of the invention. See generally, U.S. Pat. Nos.5,605,793, 5,811,238, 5,830,721, 5,834,252, and 5,837,458, and Patten,P. A., et al., Curr. Opinion Biotechnol. 8:724-33 (1997); Harayama, S.Trends Biotechnol. 16(2):76-82 (1998); Hansson, L. O., et al., J. Mol.Biol. 287:265-76 (1999); and Lorenzo, M. M. and Blasco, R. Biotechniques24(2):308-13 (1998) (each of these patents and publications are herebyincorporated by reference). In one embodiment, alteration ofpolynucleotides and corresponding polypeptides of the invention may beachieved by DNA shuffling. DNA shuffling involves the assembly of two ormore DNA segments into a desired polynucleotide sequence of theinvention molecule by homologous, or site-specific, recombination. Inanother embodiment, polynucleotides and corresponding polypeptides ofthe invention may be altered by being subjected to random mutagenesis byerror-prone PCR, random nucleotide insertion or other methods prior torecombination. In another embodiment, one or more components, motifs,sections, parts, domains, fragments, etc., of the polypeptides of theinvention may be recombined with one or more components, motifs,sections, parts, domains, fragments, etc. of one or more heterologousmolecules. In preferred embodiments, the heterologous molecules arefamily members. In further preferred embodiments, the heterologousmolecule is a growth factor such as, for example, platelet-derivedgrowth factor (PDGF), insulin-like growth factor (IGF-I), transforminggrowth factor (TGF)-alpha, epidermal growth factor (EGF), fibroblastgrowth factor (FGF), TGF-beta, bone morphogenetic protein (BMP)-2,BMP-4, BMP-5, BMP-6, BMP-7, activins A and B, decapentaplegic(dpp), 60A,OP-2, dorsalin, growth differentiation factors (GDFs), nodal, MIS,inhibin-alpha, TGF-betal, TGF-beta2, TGF-beta3, TGF-beta5, andglial-derived neurotrophic factor (GDNF).

Other preferred fragments are biologically active fragments of thepolypeptides of the invention. Biologically active fragments are thoseexhibiting activity similar, but not necessarily identical, to anactivity of the polypeptide. The biological activity of the fragmentsmay include an improved desired activity, or a decreased undesirableactivity.

Additionally, this invention provides a method of screening compounds toidentify those which modulate the action of the polypeptide of thepresent invention. An example of such an assay comprises combining amammalian fibroblast cell, a the polypeptide of the present invention,the compound to be screened and 3[H] thymidine under cell cultureconditions where the fibroblast cell would normally proliferate. Acontrol assay may be performed in the absence of the compound to bescreened and compared to the amount of fibroblast proliferation in thepresence of the compound to determine if the compound stimulatesproliferation by determining the uptake of 3[H] thymidine in each case.The amount of fibroblast cell proliferation is measured by liquidscintillation chromatography which measures the incorporation of 3[H]thymidine. Both agonist and antagonist compounds may be identified bythis procedure.

In another method, a mammalian cell or membrane preparation expressing areceptor for a polypeptide of the present invention is incubated with alabeled polypeptide of the present invention in the presence of thecompound. The ability of the compound to enhance or block thisinteraction could then be measured. Alternatively, the response of aknown second messenger system following interaction of a compound to bescreened and the receptor is measured and the ability of the compound tobind to the receptor and elicit a second messenger response is measuredto determine if the compound is a potential agonist or antagonist. Suchsecond messenger systems include but are not limited to, cAMP guanylatecyclase, ion channels or phosphoinositide hydrolysis.

All of these above assays can be used as diagnostic or prognosticmarkers. The molecules discovered using these assays can be used totreat, prevent, and/or diagnose disease or to bring about a particularresult in a patient (e.g., blood vessel growth) by activating orinhibiting the polypeptide/molecule. Moreover, the assays can discoveragents which may inhibit or enhance the production of the polypeptidesof the invention from suitably manipulated cells or tissues. Therefore,the invention includes a method of identifying compounds which bind tothe polypeptides of the invention comprising the steps of: (a)incubating a candidate binding compound with the polypeptide; and (b)determining if binding has occurred. Moreover, the invention includes amethod of identifying agonists/antagonists comprising the steps of: (a)incubating a candidate compound with the polypeptide, (b) assaying abiological activity, and (b) determining if a biological activity of thepolypeptide has been altered.

Also, one could identify molecules bind a polypeptide of the inventionexperimentally by using the beta-pleated sheet regions contained in thepolypeptide sequence of the protein. Accordingly, specific embodimentsof the invention are directed to polynucleotides encoding polypeptideswhich comprise, or alternatively consist of, the amino acid sequence ofeach beta pleated sheet regions in a disclosed polypeptide sequence.Additional embodiments of the invention are directed to polynucleotidesencoding polypeptides which comprise, or alternatively consist of, anycombination or all of contained in the polypeptide sequences of theinvention. Additional preferred embodiments of the invention aredirected to polypeptides which comprise, or alternatively consist of,the amino acid sequence of each of the beta pleated sheet regions in oneof the polypeptide sequences of the invention. Additional embodiments ofthe invention are directed to polypeptides which comprise, oralternatively consist of, any combination or all of the beta pleatedsheet regions in one of the polypeptide sequences of the invention.

Targeted Delivery

In another embodiment, the invention provides a method of deliveringcompositions to targeted cells expressing a receptor for a polypeptideof the invention, or cells expressing a cell bound form of a polypeptideof the invention.

As discussed herein, polypeptides or antibodies of the invention may beassociated with heterologous polypeptides, heterologous nucleic acids,toxins, or prodrugs via hydrophobic, hydrophilic, ionic and/or covalentinteractions. In one embodiment, the invention provides a method for thespecific delivery of compositions of the invention to cells byadministering polypeptides of the invention (including antibodies) thatare associated with heterologous polypeptides or nucleic acids. In oneexample, the invention provides a method for delivering a therapeuticprotein into the targeted cell. In another example, the inventionprovides a method for delivering a single stranded nucleic acid (e.g.,antisense or ribozymes) or double stranded nucleic acid (e.g., DNA thatcan integrate into the cell's genome or replicate episomally and thatcan be transcribed) into the targeted cell.

In another embodiment, the invention provides a method for the specificdestruction of cells (e.g., the destruction of tumor cells) byadministering polypeptides of the invention (e.g., polypeptides of theinvention or antibodies of the invention) in association with toxins orcytotoxic prodrugs.

By “toxin” is meant compounds that bind and activate endogenouscytotoxic effector systems, radioisotopes, holotoxins, modified toxins,catalytic subunits of toxins, or any molecules or enzymes not normallypresent in or on the surface of a cell that under defined conditionscause the cell's death. Toxins that may be used according to the methodsof the invention include, but are not limited to, radioisotopes known inthe art, compounds such as, for example, antibodies (or complementfixing containing portions thereof) that bind an inherent or inducedendogenous cytotoxic effector system, thymidine kinase, endonuclease,RNAse, alpha toxin, ricin, abrin, Pseudomonas exotoxin A, diphtheriatoxin, saporin, momordin, gelonin, pokeweed antiviral protein,alpha-sarcin and cholera toxin. By “cytotoxic prodrug” is meant anon-toxic compound that is converted -by an enzyme, normally present inthe cell, into a cytotoxic compound. Cytotoxic prodrugs that may be usedaccording to the methods of the invention include, but are not limitedto, glutamyl derivatives of benzoic acid mustard alkylating agent,phosphate derivatives of etoposide or mitomycin C, cytosine arabinoside,daunorubisin, and phenoxyacetamide derivatives of doxorubicin.

Drug Screening

Further contemplated is the use of the polypeptides of the presentinvention, or the polynucleotides encoding these polypeptides, to screenfor molecules which modify the activities of the polypeptides of thepresent invention. Such a method would include contacting thepolypeptide of the present invention with a selected compound(s)suspected of having antagonist or agonist activity, and assaying theactivity of these polypeptides following binding.

This invention is particularly useful for screening therapeuticcompounds by using the polypeptides of the present invention, or bindingfragments thereof, in any of a variety of drug screening techniques. Thepolypeptide or fragment employed in such a test may be affixed to asolid support, expressed on a cell surface, free in solution, or locatedintracellularly. One method of drug screening utilizes eukaryotic orprokaryotic host cells which are stably transformed with recombinantnucleic acids expressing the polypeptide or fragment. Drugs are screenedagainst such transformed cells in competitive binding assays. One maymeasure, for example, the formulation of complexes between the agentbeing tested and a polypeptide of the present invention.

Thus, the present invention provides methods of screening for drugs orany other agents which affect activities mediated by the polypeptides ofthe present invention. These methods comprise contacting such an agentwith a polypeptide of the present invention or a fragment thereof andassaying for the presence of a complex between the agent and thepolypeptide or a fragment thereof, by methods well known in the art. Insuch a competitive binding assay, the agents to screen are typicallylabeled. Following incubation, free agent is separated from that presentin bound form, and the amount of free or uncomplexed label is a measureof the ability of a particular agent to bind to the polypeptides of thepresent invention.

Another technique for drug screening provides high throughput screeningfor compounds having suitable binding affinity to the polypeptides ofthe present invention, and is described in great detail in EuropeanPatent Application 84/03564, published on Sep. 13, 1984, which isincorporated herein by reference herein. Briefly stated, large numbersof different small peptide test compounds are synthesized on a solidsubstrate, such as plastic pins or some other surface. The peptide testcompounds are reacted with polypeptides of the present invention andwashed. Bound polypeptides are then detected by methods well known inthe art. Purified polypeptides are coated directly onto plates for usein the aforementioned drug screening techniques. In addition,non-neutralizing antibodies may be used to capture the peptide andimmobilize it on the solid support.

This invention also contemplates the use of competitive drug screeningassays in which neutralizing antibodies capable of binding polypeptidesof the present invention specifically compete with a test compound forbinding to the polypeptides or fragments thereof. In this manner, theantibodies are used to detect the presence of any peptide which sharesone or more antigenic epitopes with a polypeptide of the invention.

The human HLLRCR-1 polypeptides and/or peptides of the presentinvention, or immunogenic fragments or oligopeptides thereof, can beused for screening therapeutic drugs or compounds in a variety of drugscreening techniques. The fragment employed in such a screening assaymay be free in solution, affixed to a solid support, borne on a cellsurface, or located intracellularly. The reduction or abolition ofactivity of the formation of binding complexes between the ion channelprotein and the agent being tested can be measured. Thus, the presentinvention provides a method for screening or assessing a plurality ofcompounds for their specific binding affinity with a HLLRCR-1polypeptide, or a bindable peptide fragment, of this invention,comprising providing a plurality of compounds, combining the HLLRCR-1polypeptide, or a bindable peptide fragment, with each of a plurality ofcompounds for a time sufficient to allow binding under suitableconditions and detecting binding of the HLLRCR-1 polypeptide or peptideto each of the plurality of test compounds, thereby identifying thecompounds that specifically bind to the HLLRCR-1 polypeptide or peptide.

Methods of identifying compounds that modulate the activity of the novelhuman HLLRCR-1 polypeptides and/or peptides are provided by the presentinvention and comprise combining a potential or candidate compound ordrug modulator of leucine-rich repeat containing protein biologicalactivity with an HLLRCR-1 polypeptide or peptide, for example, theHLLRCR-1 amino acid sequence as set forth in SEQ ID NO:2 and/or 4, andmeasuring an effect of the candidate compound or drug modulator on thebiological activity of the HLLRCR-1 polypeptide or peptide. Suchmeasurable effects include, for example, physical binding interaction;the ability to cleave a suitable leucine-rich repeat containing proteinsubstrate; effects on native and cloned HLLRCR-1-expressing cell line;and effects of modulators or other leucine-rich repeat containingprotein-mediated physiological measures.

Another method of identifying compounds that modulate the biologicalactivity of the novel HLLRCR-1 polypeptides of the present inventioncomprises combining a potential or candidate compound or drug modulatorof a leucine-rich repeat containing protein biological activity with ahost cell that expresses the HLLRCR-1 polypeptide and measuring aneffect of the candidate compound or drug modulator on the biologicalactivity of the HLLRCR-1 polypeptide. The host cell can also be capableof being induced to express the HLLRCR-1 polypeptide, e.g., viainducible expression. Physiological effects of a given modulatorcandidate on the HLLRCR-1 polypeptide can also be measured. Thus,cellular assays for particular leucine-rich repeat containing proteinmodulators may be either direct measurement or quantification of thephysical biological activity of the HLLRCR-1 polypeptide, or they may bemeasurement or quantification of a physiological effect. Such methodspreferably employ a HLLRCR-1 polypeptide as described herein, or anoverexpressed recombinant HLLRCR-1 polypeptide in suitable host cellscontaining an expression vector as described herein, wherein theHLLRCR-1 polypeptide is expressed, overexpressed, or undergoesupregulated expression.

Another aspect of the present invention embraces a method of screeningfor a compound that is capable of modulating the biological activity ofa HLLRCR-1 polypeptide, comprising providing a host cell containing anexpression vector harboring a nucleic acid sequence encoding a HLLRCR-1polypeptide, or a functional peptide or portion thereof (e.g., SEQ IDNOS:2); determining the biological activity of the expressed HLLRCR-1polypeptide in the absence of a modulator compound; contacting the cellwith the modulator compound and determining the biological activity ofthe expressed HLLRCR-1 polypeptide in the presence of the modulatorcompound. In such a method, a difference between the activity of theHLLRCR-1 polypeptide in the presence of the modulator compound and inthe absence of the modulator compound indicates a modulating effect ofthe compound.

Essentially any chemical compound can be employed as a potentialmodulator or ligand in the assays according to the present invention.Compounds tested as leucine-rich repeat containing protein modulatorscan be any small chemical compound, or biological entity (e.g., protein,sugar, nucleic acid, lipid). Test compounds will typically be smallchemical molecules and peptides. Generally, the compounds used aspotential modulators can be dissolved in aqueous or organic (e.g.,DMSO-based) solutions. The assays are designed to screen large chemicallibraries by automating the assay steps and providing compounds from anyconvenient source. Assays are typically run in parallel, for example, inmicrotiter formats on microtiter plates in robotic assays. There aremany suppliers of chemical compounds, including Sigma (St. Louis, Mo.),Aldrich (St. Louis, Mo.), Sigma-Aldrich (St. Louis, Mo.), FlukaChemika-Biochemica Analytika (Buchs, Switzerland), for example. Also,compounds may be synthesized by methods known in the art.

High throughput screening methodologies are particularly envisioned forthe detection of modulators of the novel HLLRCR-1 polynucleotides andpolypeptides described herein. Such high throughput screening methodstypically involve providing a combinatorial chemical or peptide librarycontaining a large number of potential therapeutic compounds (e.g.,ligand or modulator compounds). Such combinatorial chemical libraries orligand libraries are then screened in one or more assays to identifythose library members (e.g., particular chemical species or subclasses)that display a desired characteristic activity. The compounds soidentified can serve as conventional lead compounds, or can themselvesbe used as potential or actual therapeutics.

A combinatorial chemical library is a collection of diverse chemicalcompounds generated either by chemical synthesis or biologicalsynthesis, by combining a number of chemical building blocks (i.e.,reagents such as amino acids). As an example, a linear combinatoriallibrary, e.g., a polypeptide or peptide library, is formed by combininga set of chemical building blocks in every possible way for a givencompound length (i.e., the number of amino acids in a polypeptide orpeptide compound). Millions of chemical compounds can be synthesizedthrough such combinatorial mixing of chemical building blocks.

The preparation and screening of combinatorial chemical libraries iswell known to those having skill in the pertinent art. Combinatoriallibraries include, without limitation, peptide libraries (e.g. U.S. Pat.No. 5,010,175; Furka, 1991, Int. J. Pept. Prot. Res., 37:487-493; andHoughton et al., 1991, Nature, 354:84-88). Other chemistries forgenerating chemical diversity libraries can also be used. Nonlimitingexamples of chemical diversity library chemistries include, peptides(PCT Publication No. WO 91/019735), encoded peptides (PCT PublicationNo. WO 93/20242), random bio-oligomers (PCT Publication No. WO92/00091), benzodiazepines (U.S. Pat. No. 5,288,514), diversomers suchas hydantoins, benzodiazepines and dipeptides (Hobbs et al., 1993, Proc.Natl. Acad Sci. USA, 90:6909-6913), vinylogous polypeptides (Hagihara etal., 1992, J. Amer. Chem. Soc., 114:6568), nonpeptidal peptidomimeticswith glucose scaffolding (Hirschmann et al., 1992, J. Amer. Chem. Soc.,114:9217-9218), analogous organic synthesis of small compound libraries(Chen et al., 1994, J. Amer. Chem. Soc., 116:2661), oligocarbamates (Choet al., 1993, Science, 261:1303), and/or peptidyl phosphonates (Campbellet al., 1994, J. Org. Chem., 59:658), nucleic acid libraries (seeAusubel, Berger and Sambrook, all supra), peptide nucleic acid libraries(U.S. Pat. No. 5,539,083), antibody libraries (e.g., Vaughn et al.,1996, Nature Biotechnology, 14(3):309-314) and PCT/US96/10287),carbohydrate libraries (e.g., Liang et al., 1996, Science,274-1520-1522) and U.S. Pat. No. 5,593,853), small organic moleculelibraries (e.g., benzodiazepines, Baum C&EN, Jan. 18, 1993, page 33; andU.S. Pat. No. 5,288,514; isoprenoids, U.S. Pat. No. 5,569,588;thiazolidinones and metathiazanones, U.S. Pat. No. 5,549,974;pyrrolidines, U.S. Pat. Nos. 5,525,735 and 5,519,134; morpholinocompounds, U.S. Pat. No. 5,506,337; and the like).

Devices for the preparation of combinatorial libraries are commerciallyavailable (e.g., 357 MPS, 390 MPS, Advanced Chem Tech, Louisville Ky.;Symphony, Rainin, Woburn, Mass.; 433A Applied Biosystems, Foster City,Calif.; 9050 Plus, Millipore, Bedford, Mass.). In addition, a largenumber of combinatorial libraries are commercially available (e.g.,ComGenex, Princeton, N.J.; Asinex, Moscow, Russia; Tripos, Inc., St.Louis, Mo.; ChemStar, Ltd., Moscow, Russia; 3D Pharmaceuticals, Exton,Pa.; Martek Biosciences, Columbia, Md., and the like).

In one embodiment, the invention provides solid phase based in vitroassays in a high throughput format, where the cell or tissue expressingan ion channel is attached to a solid phase substrate. In such highthroughput assays, it is possible to screen up to several thousanddifferent modulators or ligands in a single day. In particular, eachwell of a microtiter plate can be used to perform a separate assayagainst a selected potential modulator, or, if concentration orincubation time effects are to be observed, every 5-10 wells can test asingle modulator. Thus, a single standard microtiter plate can assayabout 96 modulators. If 1536 well plates are used, then a single platecan easily assay from about 100 to about 1500 different compounds. It ispossible to assay several different plates per day; thus, for example,assay screens for up to about 6,000-20,000 different compounds arepossible using the described integrated systems.

In another of its aspects, the present invention encompasses screeningand small molecule (e.g., drug) detection assays which involve thedetection or identification of small molecules that can bind to a givenprotein, i.e., a HLLRCR-1 polypeptide or peptide. Particularly preferredare assays suitable for high throughput screening methodologies.

In such binding-based detection, identification, or screening assays, afunctional assay is not typically required. All that is needed is atarget protein, preferably substantially purified, and a library orpanel of compounds (e.g., ligands, drugs, small molecules) or biologicalentities to be screened or assayed for binding to the protein target.Preferably, most small molecules that bind to the target protein willmodulate activity in some manner, due to preferential, higher affinitybinding to functional areas or sites on the protein.

An example of such an assay is the fluorescence based thermal shiftassay (3-Dimensional Pharmaceuticals, Inc., 3DP, Exton, Pa.) asdescribed in U.S. Pat. Nos. 6,020,141 and 6,036,920 to Pantoliano etal.; see also, J. Zimmerman, 2000, Gen. Eng. News, 20(8)). The assayallows the detection of small molecules (e.g., drugs, ligands) that bindto expressed, and preferably purified, ion channel polypeptide based onaffinity of binding determinations by analyzing thermal unfolding curvesof protein-drug or ligand complexes. The drugs or binding moleculesdetermined by this technique can be further assayed, if desired, bymethods, such as those described herein, to determine if the moleculesaffect or modulate function or activity of the target protein.

To purify a HLLRCR-1 polypeptide or peptide to measure a biologicalbinding or ligand binding activity, the source may be a whole celllysate that can be prepared by successive freeze-thaw cycles (e.g., oneto three) in the presence of standard protease inhibitors. The HLLRCR-1polypeptide may be partially or completely purified by standard proteinpurification methods, e.g., affinity chromatography using specificantibody described infra, or by ligands specific for an epitope tagengineered into the recombinant HLLRCR-1 polypeptide molecule, also asdescribed herein. Binding activity can then be measured as described.

Compounds which are identified according to the methods provided herein,and which modulate or regulate the biological activity or physiology ofthe HLLRCR-1 polypeptides according to the present invention are apreferred embodiment of this invention. It is contemplated that suchmodulatory compounds may be employed in treatment and therapeuticmethods for treating a condition that is mediated by the novel HLLRCR-1polypeptides by administering to an individual in need of such treatmenta therapeutically effective amount of the compound identified by themethods described herein.

In addition, the present invention provides methods for treating anindividual in need of such treatment for a disease, disorder, orcondition that is mediated by the HLLRCR-1 polypeptides of theinvention, comprising administering to the individual a therapeuticallyeffective amount of the HLLRCR-1-modulating compound identified by amethod provided herein.

Antisense And Ribozyme (Antagonists)

In specific embodiments, antagonists according to the present inventionare nucleic acids corresponding to the sequences contained in SEQ IDNO:1 and/or 3, or the complementary strand thereof, nucleic acidscorresponding to the sequences contained in SEQ ID NO:3, or thecomplementary strand thereof, and/or to nucleotide sequences contained adeposited clone. In one embodiment, antisense sequence is generatedinternally by the organism, in another embodiment, the antisensesequence is separately administered (see, for example, O'Connor,Neurochem., 56:560 (1991). Oligodeoxynucleotides as Antisense Inhibitorsof Gene Expression, CRC Press, Boca Raton, Fla. (1988). Antisensetechnology can be used to control gene expression through antisense DNAor RNA, or through triple-helix formation. Antisense techniques arediscussed for example, in Okano, Neurochem., 56:560 (1991);Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRCPress, Boca Raton, Fla. (1988). Triple helix formation is discussed in,for instance, Lee et al., Nucleic Acids Research, 6:3073 (1979); Cooneyet al., Science, 241:456 (1988); and Dervan et al., Science, 251:1300(1991). The methods are based on binding of a polynucleotide to acomplementary DNA or RNA.

For example, the use of c-myc and c-myb antisense RNA constructs toinhibit the growth of the non-lymphocytic leukemia cell line HL-60 andother cell lines was previously described. (Wickstrom et al. (1988);Anfossi et al. (1989)). These experiments were performed in vitro byincubating cells with the oligoribonucleotide. A similar procedure forin vivo use is described in WO 91/15580. Briefly, a pair ofoligonucleotides for a given antisense RNA is produced as follows: Asequence complimentary to the first 15 bases of the open reading frameis flanked by an EcoR1 site on the 5 end and a HindIII site on the 3end. Next, the pair of oligonucleotides is heated at 90° C. for oneminute and then annealed in 2× ligation buffer (20 mM TRIS HCl pH 7.5,10 mM MgCl2, 10 MM dithiothreitol (DTT) and 0.2 mM ATP) and then ligatedto the EcoR1/Hind III site of the retroviral vector PMV7 (WO 91/15580).

For example, the 5′ coding portion of a polynucleotide that encodes themature polypeptide of the present invention may be used to design anantisense RNA oligonucleotide of from about 10 to 40 base pairs inlength. A DNA oligonucleotide is designed to be complementary to aregion of the gene involved in transcription thereby preventingtranscription and the production of the receptor. The antisense RNAoligonucleotide hybridizes to the mRNA in vivo and blocks translation ofthe mRNA molecule into receptor polypeptide.

In one embodiment, the antisense nucleic acid of the invention isproduced intracellularly by transcription from an exogenous sequence.For example, a vector or a portion thereof, is transcribed, producing anantisense nucleic acid (RNA) of the invention. Such a vector wouldcontain a sequence encoding the antisense nucleic acid of the invention.Such a vector can remain episomal or become chromosomally integrated, aslong as it can be transcribed to produce the desired antisense RNA. Suchvectors can be constructed by recombinant DNA technology methodsstandard in the art. Vectors can be plasmid, viral, or others known inthe art, used for replication and expression in vertebrate cells.Expression of the sequence encoding a polypeptide of the invention, orfragments thereof, can be by any promoter known in the art to act invertebrate, preferably human cells. Such promoters can be inducible orconstitutive. Such promoters include, but are not limited to, the SV40early promoter region (Bemoist and Chambon, Nature, 29:304-310 (1981),the promoter contained in the 3′ long terminal repeat of Rous sarcomavirus (Yamamoto et al., Cell, 22:787-797 (1980), the herpes thymidinepromoter (Wagner et al., Proc. Natl. Acad. Sci. U.S.A., 78:1441-1445(1981), the regulatory sequences of the metallothionein gene (Brinsteret al., Nature, 296:39-42 (1982)), etc.

The antisense nucleic acids of the invention comprise a sequencecomplementary to at least a portion of an RNA transcript of a gene ofinterest. However, absolute complementarity, although preferred, is notrequired. A sequence “complementary to at least a portion of an RNA,”referred to herein, means a sequence having sufficient complementarityto be able to hybridize with the RNA, forming a stable duplex; in thecase of double stranded antisense nucleic acids of the invention, asingle strand of the duplex DNA may thus be tested, or triplex formationmay be assayed. The ability to hybridize will depend on both the degreeof complementarity and the length of the antisense nucleic acidGenerally, the larger the hybridizing nucleic acid, the more basemismatches with a RNA sequence of the invention it may contain and stillform a stable duplex (or triplex as the case may be). One skilled in theart can ascertain a tolerable degree of mismatch by use of standardprocedures to determine the melting point of the hybridized complex.

Oligonucleotides that are complementary to the 5′ end of the message,e.g., the 5′ untranslated sequence up to and including the AUGinitiation codon, should work most efficiently at inhibitingtranslation. However, sequences complementary to the 3′ untranslatedsequences of mRNAs have been shown to be effective at inhibitingtranslation of mRNAs as well. See generally, Wagner, R., Nature,372:333-335 (1994). Thus, oligonucleotides complementary to either the5′- or 3′-non-translated, non-coding regions of a polynucleotidesequence of the invention could be used in an antisense approach toinhibit translation of endogenous mRNA. Oligonucleotides complementaryto the 5′ untranslated region of the mRNA should include the complementof the AUG start codon. Antisense oligonucleotides complementary to mRNAcoding regions are less efficient inhibitors of translation but could beused in accordance with the invention. Whether designed to hybridize tothe 5′-, 3′- or coding region of mRNA, antisense nucleic acids should beat least six nucleotides in length, and are preferably oligonucleotidesranging from 6 to about 50 nucleotides in length. In specific aspectsthe oligonucleotide is at least 10 nucleotides, at least 17 nucleotides,at least 25 nucleotides or at least 50 nucleotides.

The polynucleotides of the invention can be DNA or RNA or chimericmixtures or derivatives or modified versions thereof, single-stranded ordouble-stranded. Antisense oligonucleotides may be single or doublestranded. Double stranded RNA's may be designed based upon the teachingsof Paddison et al., Proc. Nat. Acad. Sci., 99:1443-1448 (2002); andInternational Publication Nos. WO 01/29058, and WO 99/32619; which arehereby incorporated herein by reference. The oligonucleotide can bemodified at the base moiety, sugar moiety, or phosphate backbone, forexample, to improve stability of the molecule, hybridization, etc. Theoligonucleotide may include other appended groups such as peptides(e.g., for targeting host cell receptors in vivo), or agentsfacilitating transport across the cell membrane (see, e.g., Letsinger etal., Proc. Natl. Acad. Sci. U.S.A. 86:6553-6556 (1989); Lemaitre et al.,Proc. Natl. Acad. Sci., 84:648-652 (1987); PCT Publication NO:WO88/09810, published Dec. 15, 1988) or the blood-brain barrier (see,e.g., PCT Publication NO: WO89/10134, published Apr. 25, 1988),hybridization-triggered cleavage agents. (See, e.g., Krol et al.,BioTechniques, 6:958-976 (1988)) or intercalating agents. (See, e.g.,Zon, Pharm. Res., 5:539-549 (1988)). To this end, the oligonucleotidemay be conjugated to another molecule, e.g., a peptide, hybridizationtriggered cross-linking agent, transport agent, hybridization-triggeredcleavage agent, etc.

The antisense oligonucleotide may comprise at least one modified basemoiety which is selected from the group including, but not limited to,5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,hypoxanthine, xanthine, 4-acetylcytosine,5-(carboxyhydroxylmethyl)uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine.

The antisense oligonucleotide may also comprise at least one modifiedsugar moiety selected from the group including, but not limited to,arabinose, 2-fluoroarabinose, xylulose, and hexose.

In yet another embodiment, the antisense oligonucleotide comprises atleast one modified phosphate backbone selected from the group including,but not limited to, a phosphorothioate, a phosphorodithioate, aphosphoramidothioate, a phosphoramidate, a phosphordiamidate, amethylphosphonate, an alkyl phosphotriester, and a formacetal or analogthereof.

In yet another embodiment, the antisense oligonucleotide is ana-anomeric oligonucleotide. An a-anomeric oligonucleotide forms specificdouble-stranded hybrids with complementary RNA in which, contrary to theusual b-units, the strands run parallel to each other (Gautier et al.,Nucl. Acids Res., 15:6625-6641 (1987)). The oligonucleotide is a2-0-methylribonucleotide (Inoue et al., Nucl. Acids Res., 15:6131-6148(1987)), or a chimeric RNA-DNA analogue (Inoue et al., FEBS Lett.215:327-330 (1987)).

Polynucleotides of the invention may be synthesized by standard methodsknown in the art, e.g. by use of an automated DNA synthesizer (such asare commercially available from Biosearch, Applied Biosystems, etc.). Asexamples, phosphorothioate oligonucleotides may be synthesized by themethod of Stein et al. (Nucl. Acids Res., 16:3209 (1988)),methylphosphonate oligonucleotides can be prepared by use of controlledpore glass polymer supports (Sarin et al., Proc. Natl. Acad. Sci.U.S.A., 85:7448-7451 (1988)), etc.

While antisense nucleotides complementary to the coding region sequenceof the invention could be used, those complementary to the transcribeduntranslated region are most preferred.

Potential antagonists according to the invention also include catalyticRNA, or a ribozyme (See, e.g., PCT International Publication WO90/11364, published Oct. 4, 1990; Sarver et al, Science, 247:1222-1225(1990). While ribozymes that cleave mRNA at site specific recognitionsequences can be used to destroy mRNAs corresponding to thepolynucleotides of the invention, the use of hammerhead ribozymes ispreferred. Hammerhead ribozymes cleave mRNAs at locations dictated byflanking regions that form complementary base pairs with the targetmRNA. The sole requirement is that the target mRNA have the followingsequence of two bases: 5′-UG-3′. The construction and production ofhammerhead ribozymes is well known in the art and is described morefully in Haseloff and Gerlach, Nature, 334:585-591 (1988). There arenumerous potential hammerhead ribozyme cleavage sites within eachnucleotide sequence disclosed in the sequence listing. Preferably, theribozyme is engineered so that the cleavage recognition site is locatednear the 5′ end of the mRNA corresponding to the polynucleotides of theinvention; i.e., to increase efficiency and minimize the intracellularaccumulation of non-functional mRNA transcripts.

As in the antisense approach, the ribozymes of the invention can becomposed of modified oligonucleotides (e.g. for improved stability,targeting, etc.) and should be delivered to cells which express thepolynucleotides of the invention in vivo. DNA constructs encoding theribozyme may be introduced into the cell in the same manner as describedabove for the introduction of antisense encoding DNA. A preferred methodof delivery involves using a DNA construct “encoding” the ribozyme underthe control of a strong constitutive promoter, such as, for example, polIII or pol II promoter, so that transfected cells will producesufficient quantities of the ribozyme to destroy endogenous messages andinhibit translation. Since ribozymes unlike antisense molecules, arecatalytic, a lower intracellular concentration is required forefficiency.

Antagonist/agonist compounds may be employed to inhibit the cell growthand proliferation effects of the polypeptides of the present inventionon neoplastic cells and tissues, i.e. stimulation of angiogenesis oftumors, and, therefore, retard or prevent abnormal cellular growth andproliferation, for example, in tumor formation or growth.

The antagonist/agonist may also be employed to prevent hyper-vasculardiseases, and prevent the proliferation of epithelial lens cells afterextracapsular cataract surgery. Prevention of the mitogenic activity ofthe polypeptides of the present invention may also be desirous in casessuch as restenosis after balloon angioplasty.

The antagonist/agonist may also be employed to prevent the growth ofscar tissue during wound healing.

The antagonist/agonist may also be employed to treat, prevent, and/ordiagnose the diseases described herein.

Thus, the invention provides a method of treating or preventingdiseases, disorders, and/or conditions, including but not limited to thediseases, disorders, and/or conditions listed throughout thisapplication, associated with overexpression of a polynucleotide of thepresent invention by administering to a patient (a) an antisensemolecule directed to the polynucleotide of the present invention, and/or(b) a ribozyme directed to the polynucleotide of the present invention.

Biotic Associations

A polynucleotide or polypeptide and/or agonist or antagonist of thepresent invention may increase the organisms ability, either directly orindirectly, to initiate and/or maintain biotic associations with otherorganisms. Such associations may be symbiotic, nonsymbiotic,endosymbiotic, macrosymbiotic, and/or microsymbiotic in nature. Ingeneral, a polynucleotide or polypeptide and/or agonist or antagonist ofthe present invention may increase the organisms ability to form bioticassociations with any member of the fungal, bacterial, lichen,mycorrhizal, cyanobacterial, dinoflaggellate, and/or algal, kingdom,phylums, families, classes, genuses, and/or species.

The mechanism by which a polynucleotide or polypeptide and/or agonist orantagonist of the present invention may increase the host organismsability, either directly or indirectly, to initiate and/or maintainbiotic associations is variable, though may include, modulatingosmolarity to desirable levels for the symbiont, modulating pH todesirable levels for the symbiont, modulating secretions of organicacids, modulating the secretion of specific proteins, phenoliccompounds, nutrients, or the increased expression of a protein requiredfor host-biotic organisms interactions (e.g., a receptor, ligand, etc.).Additional mechanisms are known in the art and are encompassed by theinvention (see, for example, “Microbial Signalling andCommunication”eds., R. England, G. Hobbs, N. Bainton, and D. McL.Roberts, Cambridge University Press, Cambridge, (1999); which is herebyincorporated herein by reference).

In an alternative embodiment, a polynucleotide or polypeptide and/oragonist or antagonist of the present invention may decrease the hostorganisms ability to form biotic associations with another organism,either directly or indirectly. The mechanism by which a polynucleotideor polypeptide and/or agonist or antagonist of the present invention maydecrease the host organisms ability, either directly or indirectly, toinitiate and/or maintain biotic associations with another organism isvariable, though may include, modulating osmolarity to undesirablelevels, modulating pH to undesirable levels, modulating secretions oforganic acids, modulating the secretion of specific proteins, phenoliccompounds, nutrients, or the decreased expression of a protein requiredfor host-biotic organisms interactions (e.g., a receptor, ligand, etc.).Additional mechanisms are known in the art and are encompassed by theinvention (see, for example, “Microbial Signalling andCommunication”eds., R. England, G. Hobbs, N. Bainton, and D. McL.Roberts, Cambridge University Press, Cambridge, (1999); which is herebyincorporated herein by reference).

The hosts ability to maintain biotic associations with a particularpathogen has significant implications for the overall health and fitnessof the host. For example, human hosts have symbiosis with entericbacteria in their gastrointestinal tracts, particularly in the small andlarge intestine. In fact, bacteria counts in feces of the distal colonoften approach 10¹² per milliliter of feces. Examples of bowel flora inthe gastrointestinal tract are members of the Enterobacteriaceae,Bacteriodes, in addition to a-hemolytic streptococci, E. coli,Bifobacteria, Anaerobic cocci, Eubacteria, Costridia, lactobacilli, andyeasts. Such bacteria, among other things, assist the host in theassimilation of nutrients by breaking down food stuffs not typicallybroken down by the hosts digestive system, particularly in the hostsbowel. Therefore, increasing the hosts ability to maintain such a bioticassociation would help assure proper nutrition for the host.

Aberrations in the enteric bacterial population of mammals, particularlyhumans, has been associated with the following disorders: diarrhea,ileus, chronic inflammatory disease, bowel obstruction, duodenaldiverticula, biliary calculous disease, and malnutrition. Apolynucleotide or polypeptide and/or agonist or antagonist of thepresent invention are useful for treating, detecting, diagnosing,prognosing, and/or ameliorating, either directly or indirectly, and ofthe above mentioned diseases and/or disorders associated with aberrantenteric flora population.

The composition of the intestinal flora, for example, is based upon avariety of factors, which include, but are not limited to, the age,race, diet, malnutrition, gastric acidity, bile salt excretion, gutmotility, and immune mechanisms. As a result, the polynucleotides andpolypeptides, including agonists, antagonists, and fragments thereof,may modulate the ability of a host to form biotic associations byaffecting, directly or indirectly, at least one or more of thesefactors.

Although the predominate intestinal flora comprises anaerobic organisms,an underlying percentage represents aerobes (e.g., E. coli). This issignificant as such aerobes rapidly become the predominate organisms inintraabdominal infections—effectively becoming opportunistic early ininfection pathogenesis. As a result, there is an intrinsic need tocontrol aerobe populations, particularly for immune compromisedindividuals.

In a preferred embodiment, a polynucleotides and polypeptides, includingagonists, antagonists, and fragments thereof, are useful for inhibitingbiotic associations with specific enteric symbiont organisms in aneffort to control the population of such organisms.

Biotic associations occur not only in the gastrointestinal tract, butalso on an in the integument. As opposed to the gastrointestinal flora,the cutaneous flora is comprised almost equally with aerobic andanaerobic organisms. Examples of cutaneous flora are members of thegram-positive cocci (e.g., S. aureus, coagulase-negative staphylococci,micrococcus, M. sedentarius), gram-positive bacilli (e.g.,Corynebacterium species, C. minutissimum, Brevibacterium species,Propoionibacterium species, P. acnes), gram-negative bacilli (e.g.,Acinebacter species), and fungi (Pityrosporum orbiculare). Therelatively low number of flora associated with the integument is basedupon the inability of many organisms to adhere to the skin. Theorganisms referenced above have acquired this unique ability. Therefore,the polynucleotides and polypeptides of the present invention may haveuses which include modulating the population of the cutaneous flora,either directly or indirectly.

Aberrations in the cutaneous flora are associated with a number ofsignificant diseases and/or disorders, which include, but are notlimited to the following: impetigo, ecthyma, blistering distaldactulitis, pustules, folliculitis, cutaneous abscesses, pittedkeratolysis, trichomycosis axcillaris, dermatophytosis complex, axillaryodor, erthyrasma, cheesy foot odor, acne, tinea versicolor, seborrheicdermititis, and Pityrosporum folliculitis, to name a few. Apolynucleotide or polypeptide and/or agonist or antagonist of thepresent invention are useful for treating, detecting, diagnosing,prognosing, and/or ameliorating, either directly or indirectly, and ofthe above mentioned diseases and/or disorders associated with aberrantcutaneous flora population.

Additional biotic associations, including diseases and disordersassociated with the aberrant growth of such associations, are known inthe art and are encompassed by the invention. See, for example,“Infectious Disease”, Second Edition, Eds., S. L., Gorbach, J. G.,Bartlett, and N. R., Blacklow, W. B. Saunders Company, Philadelphia,(1998); which is hereby incorporated herein by reference).

Pheromones

In another embodiment, a polynucleotide or polypeptide and/or agonist orantagonist of the present invention may increase the organisms abilityto synthesize and/or release a pheromone. Such a pheromone may, forexample, alter the organisms behavior and/or metabolism.

A polynucleotide or polypeptide and/or agonist or antagonist of thepresent invention may modulate the biosynthesis and/or release ofpheromones, the organisms ability to respond to pheromones (e.g.,behaviorally, and/or metabolically), and/or the organisms ability todetect pheromones. Preferably, any of the pheromones, and/or volatilesreleased from the organism, or induced, by a polynucleotide orpolypeptide and/or agonist or antagonist of the invention havebehavioral effects on the organism.

Other Activities

The polypeptide of the present invention, as a result of the ability tostimulate vascular endothelial cell growth, may be employed in treatmentfor stimulating re-vascularization of ischemic tissues due to variousdisease conditions such as thrombosis, arteriosclerosis, and othercardiovascular conditions. These polypeptide may also be employed tostimulate angiogenesis and limb regeneration, as discussed above.

The polypeptide may also be employed for treating wounds due toinjuries, burns, post-operative tissue repair, and ulcers since they aremitogenic to various cells of different origins, such as fibroblastcells and skeletal muscle cells, and therefore, facilitate the repair orreplacement of damaged or diseased tissue.

The polypeptide of the present invention may also be employed stimulateneuronal growth and to treat, prevent, and/or diagnose neuronal damagewhich occurs in certain neuronal disorders or neuro-degenerativeconditions such as Alzheimer's disease, Parkinson's disease, andAIDS-related complex. The polypeptide of the invention may have theability to stimulate chondrocyte growth, therefore, they may be employedto enhance bone and periodontal regeneration and aid in tissuetransplants or bone grafts.

The polypeptide of the present invention may be also be employed toprevent skin aging due to sunburn by stimulating keratinocyte growth.

The polypeptide of the invention may also be employed for preventinghair loss, since FGF family members activate hair-forming cells andpromotes melanocyte growth. Along the same lines, the polypeptides ofthe present invention may be employed to stimulate growth anddifferentiation of hematopoietic cells and bone marrow cells when usedin combination with other cytokines.

The polypeptide of the invention may also be employed to maintain organsbefore transplantation or for supporting cell culture of primarytissues.

The polypeptide of the present invention may also be employed forinducing tissue of mesodermal origin to differentiate in early embryos.

The polypeptide or polynucleotides and/or agonist or antagonists of thepresent invention may also increase or decrease the differentiation orproliferation of embryonic stem cells, besides, as discussed above,hematopoietic lineage.

The polypeptide or polynucleotides and/or agonist or antagonists of thepresent invention may also be used to modulate mammaliancharacteristics, such as body height, weight, hair color, eye color,skin, percentage of adipose tissue, pigmentation, size, and shape (e.g.,cosmetic surgery). Similarly, polypeptides or polynucleotides and/oragonist or antagonists of the present invention may be used to modulatemammalian metabolism affecting catabolism, anabolism, processing,utilization, and storage of energy.

Polypeptide or polynucleotides and/or agonist or antagonists of thepresent invention may be used to change a mammal's mental state orphysical state by influencing biorhythms, caricadic rhythms, depression(including depressive diseases, disorders, and/or conditions), tendencyfor violence, tolerance for pain, reproductive capabilities (preferablyby Activin or Inhibin-like activity), hormonal or endocrine levels,appetite, libido, memory, stress, or other cognitive qualities.

Polypeptide or polynucleotides and/or agonist or antagonists of thepresent invention may also be used as a food additive or preservative,such as to increase or decrease storage capabilities, fat content,lipid, protein, carbohydrate, vitamins, minerals, cofactors or othernutritional components.

Polypeptide or polynucleotides and/or agonist or antagonists of thepresent invention may also be used to increase the efficacy of apharmaceutical composition, either directly or indirectly. Such a usemay be administered in simultaneous conjunction with saidpharmaceutical, or separately through either the same or different routeof administration (e.g., intravenous for the polynucleotide orpolypeptide of the present invention, and orally for the pharmaceutical,among others described herein.).

Polypeptide or polynucleotides and/or agonist or antagonists of thepresent invention may also be used to prepare individuals forextraterrestrial travel, low gravity environments, prolonged exposure toextraterrestrial radiation levels, low oxygen levels, reduction ofmetabolic activity, exposure to extraterrestrial pathogens, etc. Such ause may be administered either prior to an extraterrestrial event,during an extraterrestrial event, or both. Moreover, such a use mayresult in a number of beneficial changes in the recipient, such as, forexample, any one of the following, non-limiting, effects: an increasedlevel of hematopoietic cells, particularly red blood cells which wouldaid the recipient in coping with low oxygen levels; an increased levelof B-cells, T-cells, antigen presenting cells, and/or macrophages, whichwould aid the recipient in coping with exposure to extraterrestrialpathogens, for example; a temporary (i.e., reversible) inhibition ofhematopoietic cell production which would aid the recipient in copingwith exposure to extraterrestrial radiation levels; increase and/orstability of bone mass which would aid the recipient in coping with lowgravity environments; and/or decreased metabolism which wouldeffectively facilitate the recipients ability to prolong theirextraterrestrial travel by any one of the following, non-limiting means:(i) aid the recipient by decreasing their basal daily energyrequirements; (ii) effectively lower the level of oxidative and/ormetabolic stress in recipient (i.e., to enable recipient to cope withincreased extraterrestial radiation levels by decreasing the level ofinternal oxidative/metabolic damage acquired during normal basal energyrequirements; and/or (iii) enabling recipient to subsist at a lowermetabolic temperature (i.e., cryogenic, and/or sub-cryogenicenvironment).

Also preferred is a method of treatment of an individual in need of anincreased level of a protein activity, which method comprisesadministering to such an individual a pharmaceutical compositioncomprising an amount of an isolated polypeptide, polynucleotide, orantibody of the claimed invention effective to increase the level ofsaid protein activity in said individual.

Having generally described the invention, the same will be more readilyunderstood by reference to the following examples, which are provided byway of illustration and are not intended as limiting.

REFERENCES

-   Dixon, M. S., Golstein, C., Thomas, C. M., van der Biezen, E. A.,    and Jones, J. D. G. (2000). Genetic complexity of pathogen    perception by plants: The example of Rcr3, a tomato gene required    specifically by Cf-2. Proc. Natl. Acad. Sci. USA 97, 8807-8814.-   Eldon, E., Kooyer, S., D'Evelyn, D., Duman, M., Lawinger, P., Botas,    J., and Bellen, H. (1994). The Drosophila 18 wheeler is required for    morphogenesis and has striking similarities to Toll. Development    120, 885-899.-   Fournier, A. E., GrandPre, T., and Strittmatter, S. M. (2001).    Identification of a receptor mediating Nogo-66 inhibition of axonal    regeneration. Nature 409, 341-346.-   Gavrieli, Y., Sherman, Y., and Ben-Sasson, S. A. (1992).    Identification of programmed cell death in situ via specific    labeling of nuclear DNA fragmentation. J. Cell Biol. 119, 493-501.-   Halfon, M., Hashimoto, C., and Keshishian, H. (1995). The Drosophila    Toll gene functions zygotically and is necessary for proper    motoneuron and muscle development. Dev. Biol. 169, 151-167.-   Harton, J. A., and Ting, J. P. (2000). Class II transactivator:    Mastering the art of major histocompatibility complex expression.    Molecular and Cellular Biology 20, 6185-6194.-   Inohara, N., Koseki, T., Lin, J., Peso, L., Lucas, P. C., Chen, F.    F., Ogura, Y., and Nunez, G. (2000). An induced proximity model for    NF-kB activation in the Nod1/RICK and RIP signaling pathways.    Journal of Biological Chemistry 275, 27823-27831.-   Inohara, N., Koseki, T., Peso, L., Hu, Y., Yee, C., Chen, S.,    Carrio, R., Merina, J., Liu, D., Ni, J., and Nunez, G. (1999). Nodl,    an Apaf-1-like activator of caspase-9 and nuclear factor-kB. Journal    of Biological Chemistry 274, 14560-14567.-   Jacobs, J., and Goodman, C. (1989). Embryonic development of axon    pathways in the Drosophila CNS. I. A glial cell scaffold appears    before the first growth cones. J. Neurosci. 9, 2402-2411.-   Jones, D. A., McIntire, L. V., Smith, C. W., and Picker, L. J.    (1994). A two step adhesion cascade for T-cell/endothelial cell    interaction under flow conditions. J. Clin. Invest. 94, 2443-2450.-   Liang, Y., Annan, R. S., Carr, S. A., Popp, S., Mevissen, M.,    Margolis, R. K., and Margolis, R. U. (1999). Mammalina homologues of    the Drosophila slit protein are ligands of the herparan sulfate    proteoglycan-i in brain. J. Biol. Chem. 274, 17885-1792.-   Schneider, D. S., Hudson, K. L., Lin, T., and Anderson, K. V.    (1991). Dominant and recessive mutations defme fuictional domains of    Toll, a transmembrane protein required for dorsal-ventral polarity    in the Drosophila embryo. Genes and Development 5, 797-807.-   Sean, G. S., Buchanan, C., and Gay, N. J. (1996). Structural and    functional diversity in the leucine rich repeat family of proteins.    Prog. Biophys. Molec. Biol. 65, 1-44.-   van Der Voort, R., Keehnen, R. .M., Beuling, E. A., Spaargaren, M.,    and Pals, S. T. (2000). Regulation of cytokine signaling by B cell    antigen receptor and CD40-controlled expression of heparan sulfate    proteoglycans. J. Exp. Med. 192, 1115-1124.-   Verbeek, M. M., Otte-Holler, I., van den Born, J., van den    Heuvel, L. P., Wesseling, P., and M., d. W. R. (1999). Agrin is a    major heparan sulfate proteoglycan accummulating in Alzheimer's    disease brain. Am. J. Pathol. 155, 2115-2125.

EXAMPLES Description of the Preferred Embodiments Example 1Bioinformatics Analysis

G-protein coupled receptor sequences were used as probes to search thehuman genomic sequence database. The GPCR probe sequences werenon-olfactory GPCR sequences obtained through the GPCR database at EMBL(http://www.gpcr.org/7tm/). The search program used was gapped BLAST.The top genomic exon hits from the BLAST results were searched backagainst the non-redundant protein and patent sequence databases. Fromthis analysis, exons encoding potential novel GPCRs and transmembraneproteins were identified based on sequence homology. Also, the genomicregion surrounding the matching exons were analyzed. Based on thisanalysis, potential full-length sequence of a novel human transmembraneprotein, GPCR111, was identified. The genomic region extending beyondthe GPCR111 exon sequences corresponded to human bac AL139285. Thefull-length clone of this transmembrane protein was experimentallyobtained using a portion of the AL139285 genomic sequence (see SEQ IDNO:45).

Additional characterization of the GPCR111 clone led to the elucidationthat this clone encoded a novel human leucine-rich repeat (LLR) domaincontaining protein. The sequence of the clone was determined usingprocedures known in the art, and described herein, to obtain thefull-length clone encoding the novel HLRRCR-1 protein. The completeprotein sequence of this protein was analyzed for potentialtransmembrane domains. TMPRED program (5) was used for transmembraneprediction. Also, these proteins were analyzed for potential motifs andprotein domains. Motifs program in GCG (GCG is a software package fromGenetics Computer Group of Wisconsin) was used for identifying thepotential motifs in the protein. Protein domains were analyzed by usingHMMER. HMMER is a freely distributable implementation of profile HiddenMarkov Model (HMM) software for protein sequence analysis(http://hmmer.wustl.edu/). The protein domain search set used was Pfam(http://pfam.wustl.edu/). Pfam is a large collection of multiplesequence alignments and hidden Markov models of protein domains covering2478 protein families. By these analyses, the HLLRCR-1 protein has beenpredicted to comprise at least six or more leucine-rich repeat domains.

Example 2 Method for Constructing a Size Fractionated Brain and TestiscDNA Library

Brain and testis poly A+RNA was purchased from Clontech and convertedinto double stranded cDNA using the SuperScript™ Plasmid System for cDNASynthesis and Plasmid Cloning (Life Technologies) except that noradioisotope was incorporated in either of the cDNA synthesis steps andthat the cDNA was fractionated by HPLC. This was accomplished on aTransGenomics HPLC system equipped with a size exclusion column(TosoHass) with dimensions of 7.8 mm×30 cm and a particle size of 10 μm.Tris buffered saline was used as the mobile phase, and the column wasrun at a flow rate of 0.5 mL/min. The resulting chromatograms wereanalyzed to determine which fractions should be pooled to obtain thelargest cDNA's; generally fractions that eluted in the range of 12 to 15minutes were pooled. The cDNA was precipitated prior to ligation intothe Sal I/Not I sites in the pSport vector supplied with the kit. Usinga combination of PCR with primers directed to the ends of the vector andSal I/Not I restriction enzyme digestion of mini-prep DNA, it wasdetermined that the average insert size of the library was greater the3.5 Kb. The overall complexity of the library was greater than 10⁷independent clones. The library was amplified in semi-solid agar for 2days at 30° C.

An aliquot (200 microliters) of the amplified library was inoculatedinto a 200 ml culture for single-stranded DNA isolation bysuper-infection with a f1 helper phage. After overnight grow, thereleased phage particles with precipitated with PEG and the DNA isolatedwith proteinase K, SDS and phenol extractions. The single strandedcircular DNA was concentrated by ethanol precipitation and used for thecDNA capture experiments described below.

Example 3 Cloning of the Novel Human HLLRCR-1 Polynucleotide

Using a portion of the BAC AL139285 sequence (SEQ ID NO:45), thefollowing PCR primer pairs and antisense 80 bp 5′ biotinylatedoligonucleotides were designed and obtained from Genset Oligos (SanDiego, Calif.) for use in the cloning methods described below. SEQ IDOligo Name Oligonucleotide Sequence Length NO: GPCR111-sTGATCACACCTTCAGCAAGC 20 20 GPCR111-a TGCCTCCGAAAGAACTGTCT 20 21GPCR111-80b bTGAGATTAGTAATCTGATTCCCATCCAG 80 19CTGGAGCCGGGTCAGGCCGCTTGTGTTTC GGAACCAAGACCCTCGTAGGGTG

One microliter (one hundred and fifty nanograms) of a biotinylated oligo(SEQ ID NO:8) was added to six microliters (six micrograms) of a mixtureof the single-stranded covalently closed circular brain and testis cDNAlibraries described herein, and seven microliters of 100% formamide in a0.5 ml PCR tube. The mixture was heated in a thermal cycler to 95° C.for 2 mins. Fourteen microliters of 2× hybridization buffer (50%formamide, 1.5 M NaCl, 0.04 M NaPO₄, pH 7.2, 5 mM EDTA, 0.2% SDS) wasadded to the heated probe/cDNA library mixture and incubated at 42° C.for 26 hours. Hybrids between the biotinylated oligo and the circularcDNA were isolated by diluting the hybridization mixture to 220microliters in a solution containing 1 M NaCl, 10 mM Tris-HCl pH 7.5, 1mM EDTA, pH 8.0 and adding 125 microliters of streptavidin magneticbeads. This solution was incubated at 42° C. for 60 mins, mixing every 5mins to resuspend the beads. The beads were separated from the solutionwith a magnet and the beads washed three times in 200 microliters of0.1× SSPE, 0.1% SDS at 45° C.

The single stranded cDNAs were released from the biotinlyatedoligo/streptavidin magnetic bead complex by adding 50 microliters of 0.1N NaOH and incubating at room temperature for 10 mins. Six microlitersof 3 M Sodium Acetate was added along with 15 micrograms of glycogen andthe solution ethanol precipitated with 120 microliters of 100% ethanol.The DNA was resuspend in 12 microliters of TE (10 mM Tris-HCl, pH 8.0),1 mM EDTA, pH 8.0). The single stranded cDNA was converted into doublestrands in a thermal cycler by mixing 5 microliters of the captured DNAwith 1.5 microliters 10 micromolar standard SP6 primer (homologous to asequence on the cDNA cloning vector) and 1.5 microliters of 10× PCRbuffer. The mixture was heated to 95° C. for 20 seconds, then rampeddown to 59° C. At this time 15 microliters of a repair mix, that waspreheated to 70° C. (Repair mix contains 4 microliters of 5 mM dNTPs(1.25 mM each), 1.5 microliters of 10× PCR buffer, 9.25 microliters ofwater, and 0.25 microliters of Taq polymerase). The solution was rampedback to 73° C. and incubated for 23 mins. The repaired DNA was ethanolprecipitate and resuspended in 10 microliters of TE. Two microliterswere electroporated in E. coli DH12S cells and resulting colonies werescreen by PCR, using a primer pairs designed from the EST sequences toidentify the proper cDNAs. Those cDNA clones that were positive by PCRhad the inserts sized and two clones for each probe were chosen for DNAsequencing.

The full-length nucleotide sequence and the encoded polypeptide forHLLRCR-1 is shown in FIGS. 1A-C.

Example 4 Expression Profiling of the Novel Human HLLRCR-1 Polypeptide

The following PCR primer pair was used to measure the steady statelevels of HLLRCR-1 mRNA by quantitative PCR: Sense:5′-TGATCACACCTTCAGCAAGC-3′ (SEQ ID NO:2 and/or 40) Antisense:5′-TGCCTCCGAAAGAACTGTCT-3′ (SEQ ID NO:2 and/or 41)

Briefly, first strand cDNA was made from commercially available mRNA(Clontech) and subjected to real time quantitative PCR using a PE 5700instrument (Applied Biosystems, Foster City, Calif.) which detects theamount of DNA amplified during each cycle by the fluorescent output ofSYBR green, a DNA binding dye specific for double strands. The primerpairs provided above were used in the PCR reaction. The specificity ofthe primer pair for its target was verified by performing a thermaldenaturation profile at the end of the run which gave an indication ofthe number of different DNA hybridization complexes present bydetermining melting Tm. In the case of the novel HLLRCR-1 gene primerpairs, only one DNA fragment was detected having a homogeneous meltingpoint. Contributions of contaminating genomic DNA to the assessment oftissue abundance is controlled by performing the PCR with first strandcDNA made with and without reverse transcriptase. In all cases, thecontribution of material amplified in the no reverse transcriptasecontrols was negligible.

Small variations in the amount of cDNA used in each tube was determinedby performing a parallel experiment using a primer pair for a geneexpressed in equal amounts in all tissues, cyclophilin. These data wereused to normalize the data obtained with the HLLRCR-1 primer pairsdescribed herein. The PCR data was converted into a relative assessmentof the difference in transcript abundance amongst the tissues tested andthe data are presented in bar graph form in FIG. 5. As shown,transcripts corresponding to HLLRCR-1 were expressed predominately highin the heart; significantly in the testis; and to a lesser extent, inbrain, spinal cord, and brain subregions.

Example 5 Method of Assessing the Expression Profile of the NovelHLRRCR-1 Polypeptides of the Present Invention in a Variety of CancerCell Lines

RNA quantification may be performed using the Taqman® real-time-PCRfluorogenic assay. The Taqman® assay is one of the most precise methodsfor assaying the concentration of nucleic acid templates.

All cell lines were grown using standard conditions: RPMI 1640supplemented with 10% fetal bovine serum, 100 IU/ml penicillin, 100mg/ml streptomycin, and 2 mM L-glutamine, 10 mM Hepes (all fromGibcoBRL; Rockville, Md.). Eighty percent confluent cells were washedtwice with phosphate-buffered saline (GibcoBRL) and harvested using0.25% trypsin (GibcoBRL). RNA was prepared using the RNeasy Maxi Kitfrom Qiagen (Valencia, Calif.).

cDNA template for real-time PCR may be generated using the Superscript™First Strand Synthesis system for RT-PCR.

SYBR Green real-time PCR reactions were prepared as follows: Thereaction mix consisted of 20 ng first strand cDNA; 50 nM Forward Primer;50 nM Reverse Primer; 0.75× SYBR Green I (Sigma); 1× SYBR Green PCRBuffer (50 mMTris-HCl pH8.3, 7 5mM KCl); 10%DMSO; 3 mM MgCl₂; 300 μMeach DATP, dGTP, dTTP, dCTP; 1 U Platinum® Taq DNA Polymerase HighFidelity (Cat# 11304-029; Life Technologies; Rockville, Md.); 1:50dilution; ROX (Life Technologies). Real-time PCR was performed using anApplied Biosystems 5700 Sequence Detection System. Conditions were 95°C. for 10 min (denaturation and activation of Platinum® Taq DNAPolymerase), 40 cycles of PCR (95° C. for 15 sec, 60° C. for 1 min). PCRproducts are analyzed for uniform melting using an analysis algorithmbuilt into the 5700 Sequence Detection System. Forward primer: 1025GPCR111-s: 5′-TGATCACACCTTCAGCAAGC-3′; (SEQ ID NO:89) and Reverseprimer: 1026 GPCR111-a: 5′-TGCCTCCGAAAGAACTGTCT-3′ (SEQ ID NO:90)

cDNA quantification used in the normalization of template quantity wasperformed using Taqman® technology. Taqman® reactions are prepared asfollows: The reaction mix consisted of 20 ng first strand cDNA; 25 nMGAPDH-F3, Forward Primer; 250 nM GAPDH-R1 Reverse Primer; 200 nMGAPDH-PVIC Taqman® Probe (fluorescent dye labeled oligonucleotideprimer); 1× Buffer A (Applied Biosystems); 5.5 mM MgCl2; 300 μM dATP,dGTP, dTTP, dCTP; 1 U Amplitaq Gold (Applied Biosystems). GAPDH,D-glyceraldehyde-3-phosphate dehydrogenase, was used as control tonormalize mRNA levels.

Real-time PCR was performed using an Applied Biosystems 7700 SequenceDetection System. Conditions were 95° C. for 10 min. (denaturation andactivation of Amplitaq Gold), 40 cycles of PCR (95° C. for 15 sec, 60°C. for 1 min).

The sequences for the GAPDH oligonucleotides used in the Taqman®reactions are as follows: GAPDH-F3- 5′-AGCCGAGCCACATCGCT-3′ (SEQ IDNO:91) GAPDH-R1- 5′-GTGACCAGGCGCCCAATAC-3′ (SEQ ID NO:92) GAPDH-PVICTaqman ® Probe-VIC- 5′-CAAATCCGTTGACTCCGACCTTCACCTT-3′ (SEQ ID NO:93)TAMRA

The Sequence Detection System generates a Ct (threshold cycle) valuethat is used to calculate a concentration for each input cDNA template.cDNA levels for each gene of interest are normalized to GAPDH cDNAlevels to compensate for variations in total cDNA quantity in the inputsample. This is done by generating GAPDH Ct values for each cell line.Ct values for the gene of interest and GAPDH are inserted into amodified version of the δδCt equation (Applied Biosystems Prism® 7700Sequence Detection System User Bulletin #2), which is used to calculatea GAPDH normalized relative cDNA level for each specific cDNA. The δδCtequation is as follows: relative quantity of nucleic acid template=2^(δδCt)=2^((δCta-δCtb)), where δCta=Ct target−Ct GAPDH, and δCtb=Ctreference−Ct GAPDH. (No reference cell line was used for the calculationof relative quantity; δCtb was defined as 21).

The Graph # of Table 1 corresponds to the tissue type position number ofFIG. 8. Interestingly, HLRRCR-1 (also known as GPCR111) was found to beexpressed 98 and 5 fold greater in lung and colon tumor cell lines,respectively, in comparison to other cancer cell lines in the OCLP-1(oncology cell line panel). TABLE 1 Quantitative Expression DifferenceGraph # Name Tissue (GADPH vs. HLRRCR-1). 1 AIN 4 breast 1.0E+00 2 AIN4T breast 1.0E+00 3 AIN4/myc breast 1.0E+00 4 BT-20 breast 1.0E+00 5BT-474 breast 1.0E+00 6 BT-483 breast 8.4E−01 7 BT-549 breast 1.0E+00 8DU4475 breast 2.1E+01 9 H3396 breast 1.0E+00 10 HBL100 breast 1.0E+00 11Her2 MCF-7 breast 1.0E+00 12 HS 578T breast 1.0E+00 13 MCF7 breast1.0E+00 14 MCF-7/AdrR breast 1.0E+00 15 MDAH 2774 breast 1.0E+00 16MDA-MB-175-VII breast 1.0E+00 17 MDA-MB-231 breast 1.0E+00 18 MDA-MB-453breast 1.0E+00 19 MDA-MB-468 breast 1.0E+00 20 Pat-21 R60 breast ND 21SKBR3 breast 1.0E+00 22 T47D breast 1.0E+00 23 UACC-812 breast 1.0E+0024 ZR-75-1 breast 5.6E−01 25 C-33A cervical 5.6E+00 26 Ca Ski cervical1.0E+00 27 HeLa cervical 1.0E+00 28 HT-3 cervical 1.0E+00 29 ME-180cervical 1.0E+00 30 SiHa cervical 1.0E+00 31 SW756 cervical 1.0E+00 32CACO-2 colon 1.0E+00 33 CCD-112Co colon 1.0E+00 34 CCD-33Co colon1.0E+00 35 Colo 205 colon 1.5E+00 36 Colo 320DM colon 1.0E+00 37 Colo201colon 1.3E+00 38 Cx-1 colon 1.0E+00 39 ddH2O control ND 40 HCT116 colon1.0E+00 41 HCT116/epo5 colon 1.1E+00 42 HCT116/ras colon 1.5E+00 43HCT116/TX15CR colon 1.3E+01 44 HCT116/vivo colon 1.0E+00 45 HCT116/VM46colon 1.0E+00 46 HCT116/VP35 colon 4.1E+00 47 HCT-8 colon 1.0E+00 48HT-29 colon 5.3E+01 49 LoVo colon 1.2E+00 50 LS 174T colon 1.0E+00 51LS123 colon 1.0E+00 52 MIP colon 1.0E+00 53 SK-CO-1 colon 1.0E+00 54SW1417 colon 1.0E+00 55 SW403 colon 1.0E+00 56 SW480 colon 1.0E+00 57SW620 colon 1.0E+00 58 SW837 colon 1.0E+00 59 T84 colon 1.0E+00 60CCD-18Co colon, 1.0E+00 fibroblast 61 HT-1080 fibrosarcoma 1.0E+00 62CCRF-CEM leukemia 1.0E+00 63 HL-60 leukemia 1.0E+00 64 K562 leukemia5.4E+00 65 A-427 lung 1.0E+00 66 A549 lung 1.0E+00 67 Calu-3 lung1.0E+00 68 Calu-6 lung 1.0E+00 69 ChaGo-K-1 lung 1.7E+00 70 DMS 114 lung7.4E+00 71 LX-1 lung 1.0E+00 72 MRC-5 lung 1.0E+00 73 MSTO-211H lung1.0E+00 74 NCI-H596 lung 1.0E+00 75 SHP-77 lung 9.8E+02 76 Sk-LU-1 lung1.1E+00 77 SK-MES-1 lung 3.3E+00 78 SW1271 lung 1.0E+00 79 SW1573 lung1.0E+00 80 SW900 lung 1.0E+00 81 Hs 294T melanoma 1.0E+00 82 A2780/DDP-Rovarian 1.0E+00 83 A2780/DDP-S ovarian 1.0E+00 84 A2780/epo5 ovarian1.7E+00 85 A2780/TAX-R ovarian 2.3E+01 86 A2780/TAX-S ovarian 5.5E+01 87Caov-3 ovarian 1.0E+00 88 ES-2 ovarian 1.0E+00 89 HOC-76 ovarian Mouse90 OVCAR-3 ovarian 1.0E+00 91 PA-1 ovarian 1.0E+00 92 SW 626 ovarian1.0E+00 93 UPN251 ovarian 1.8E+00 94 LNCAP prostate 2.8E+00 95 PC-3prostate 1.0E+00 96 A431 squamous 1.0E+00

Example 6 Method of Assessing the Expression Profile of the NovelHLRRCR-1 Polypeptides of the Present Invention using Expanded mRNATissue and Cell Sources

Total RNA from tissues was isolated using the TriZol protocol(Invitrogen) and quantified by determining its absorbance at 260 nM. Anassessment of the 18 s and 28 s ribosomal RNA bands was made bydenaturing gel electrophoresis to determine RNA integrity.

The specific sequence to be measured was aligned with related genesfound in GenBank to identity regions of significant sequence divergenceto maximize primer and probe specificity. Gene-specific primers andprobes were designed using the ABI primer express software to amplifysmall amplicons (150 base pairs or less) to maximize the likelihood thatthe primers function at 100% efficiency. All primer/probe sequences weresearched against Public Genbank databases to ensure target specificity.Primers and probes were obtained from ABI.

For HLLRCR-1, the primer probe sequences were as follows Forward Primer5′-AGCAACATCGGCCTCTGAGT-3′ (SEQ ID NO:94) Reverse Primer5′-TGCCTACCCCACGGTCTTT-3′ (SEQ ID NO:95) TaqMan Probe5′-CAACCCTTGACGGATCATTTAGAA-3′ (SEQ ID NO:96)

DNA Contamination

To access the level of contaminating genomic DNA in the RNA, the RNA wasdivided into 2 aliquots and one half was treated with Rnase-free Dnase(Invitrogen). Samples from both the Dnase-treated and non-treated werethen subjected to reverse transcription reactions with (RT+) and without(RT−) the presence of reverse transcriptase. TaqMan assays were carriedout with gene-specific primers (see above) and the contribution ofgenomic DNA to the signal detected was evaluated by comparing thethreshold cycles obtained with the RT+/RT− non-Dnase treated RNA to thaton the RT+/RT− Dnase treated RNA. The amount of signal contributed bygenomic DNA in the Dnased RT− RNA must be less that 10% of that obtainedwith Dnased RT+RNA. If not the RNA was not used in actual experiments.

Reverse Transcription Reaction and Sequence Detection

100 ng of Dnase-treated total RNA was annealed to 2.5 μM of therespective gene-specific reverse primer in the presence of 5.5 mMMagnesium Chloride by heating the sample to 72° C. for 2 min and thencooling to 55° C. for 30 min. 1.25 U/μl of MuLv reverse transcriptaseand 500 μM of each dNTP was added to the reaction and the tube wasincubated at 37° C. for 30 min. The sample was then heated to 90° C. for5 min to denature enzyme.

Quantitative sequence detection was carried out on an ABI PRISM 7700 byadding to the reverse transcribed reaction 2.5 μM forward and reverseprimers, 2.0 μM of the TaqMan probe, 500 μM of each dNTP, buffer and 5UAmpliTaq Gold™. The PCR reaction was then held at 94° C. for 12 min,followed by 40 cycles of 94° C. for 15 sec and 60° C. for 30 sec.

Data Handling

The threshold cycle (Ct) of the lowest expressing tissue (the highest Ctvalue) was used as the baseline of expression and all other tissues wereexpressed as the relative abundance to that tissue by calculating thedifference in Ct value between the baseline and the other tissues andusing it as the exponent in 2^((ΔCt))

The expanded expression profile of the HLLRCR-1 polypeptide is providedin FIG. 9 and 10 and described elsewhere herein.

Example 7 Method of Assessing the Ability of HLLRCR-1 to ModulateApoptosis

The role of the novel HLLRCR-1 polypeptides in either promoting orinhibiting apoptosis could be determined by the generation oftransfected cell lines with the HLLRCR-1 polynucleotides of the presentinvention, either transient or stable, using methods known in the artand/or described herein, and any combination of commonly used assays forthe detection of DNA fragmentation. One representative example being theTUNEL assay (Gavrieli, Y., Sherman, Y., Ben, Sasson, S A, J. Cell,Biol., 119(3):493-501, (1992); which is hereby incorporated herein byreference in its entirety) which involves end labeling broken ends ofdouble-stranded DNA with biotin-conjugated dUTP using terminaltransferase. Cells undergoing cell death can then be easily detected bystaining with FITC-conjugated streptavidin and flow cytometricquantitation.

Alternatively, HLLRCR-1 can be expressed by transforming a mammaliancell line such as COS7, HeLa or CHO with an eukaryotic expression vectorencoding HLLRCR-1. Eukaryotic expression vectors are commerciallyavailable, and the techniques to introduce them into cells are wellknown to those skilled in the art. The cells with and without theHLLRCR-1 expression vector are incubated for 48-72 hours aftertransformation under conditions appropriate for the cell line to allowexpression of HLLRCR-1. Phase microscopy is subsequently used to comparethe mitotic index of transformed versus control cells. An increase inthe mito tic index where HLLRCR-1 stimulates cell proliferationindicates apoptotic activity. Likewise, a decrease in cell numbers whereHLLRCR-1 stimulates apoptosis indicates apoptotic activity.

The invention encompasses other assay methods known in the art and/ordescribed herein.

Example 8 Method of Assessing the Ability of HLLRCR-1 to ModulateCellular Adhesion

The role of the novel HLLRCR-1 polypeptides in promoting cell adhesionevents could be determined by the generation of transfected cell lineswith the HLLRCR-1 polynucleotides of the present invention, eithertransient or stable, and then subjected such cells to a hydrodynamicassay that can evaluate the relative importance of variousreceptor/ligand interactions in cell-cell and cell-substrate adhesion.Dynamic adhesion assays can simulate the forces found in the bloodstreamand may be used to estimate the strength of the bonds between cells andligands. Once representative assay is described by Jones et al, 1994.The skilled artisan would appreciate that this assay could readily beadapted to address the potential for HLLRCR-1 to modulate cellularadhesion.

The role of the newly described LRR protein in modulating the molecularevents involved in autoimmunity and inflammation could be assayed in avariety of ways both in vitro (cell culture models) and in vivo (animalmodels). The development of over-expressing cell lines could be used todetermine the effect of the novel LRR proteins on the induction ofTNF-α, NFκB activation and various cytokine release profiles afterstimulation with bacterial derived lipopolysaccharide (LPS).

The invention encompasses other assay methods known in the art and/ordescribed herein.

Example 9 Method of Assessing the Physiological Function of the HLLRCR-1Polypeptide at the Cellular Level

The physiological function of the HLLRCR-1 polypeptide may be assessedby expressing the sequences encoding HLLRCR-1 at physiologicallyelevated levels in mammalian cell culture systems. cDNA is subclonedinto a mammalian expression vector containing a strong promoter thatdrives high levels of cDNA expression (examples are provided elsewhereherein). Vectors of choice include pCMV SPORT (Life Technologies) andpCR3.1 (Invitrogen, Carlsbad Calif.), both of which contain thecytomegalovirus promoter. 5-10, ug of recombinant vector are transientlytransfected into a human cell line, preferably of endothelial orhematopoietic origin, using either liposome formulations orelectroporation. 1-2 ug of an additional plasmid containing sequencesencoding a marker protein are cotransfected. Expression of a markerprotein provides a means to distinguish transfected cells fromnontransfected cells and is a reliable predictor of cDNA expression fromthe recombinant vector. Marker proteins of choice include, e.g., GreenFluorescent Protein (GFP; Clontech), CD64, or a CD64-GFP fusion protein.Flow cytometry (FCM), an automated, laser optics-based technique, isused to identify transfected cells expressing GFP or CD64-GFP and toevaluate the apoptotic state of the cells and other cellular properties.FCM detects and quantifies the uptake of fluorescent molecules thatdiagnose events preceding or coincident with cell death. These eventsinclude changes in nuclear DNA content as measured by staining of DNAwith propidium iodide; changes in cell size and granularity as measuredby forward light scatter and 90 degree side light scatter;down-regulation of DNA synthesis as measured by decrease inbromodeoxyuridine uptake; alterations in expression of cell surface andintracellular proteins as measured by reactivity with specificantibodies; and alterations in plasma membrane composition as measuredby the binding of fluorescein-conjugated Annexin V protein to the cellsurface. Methods in flow cytometry are discussed in Ormerod, M. G.(1994) Flow Cvtometrv, Oxford, New York N.Y.

The influence of HLLRCR-1 polypeptides on gene expression can beassessed using highly purified populations of cells transfected withsequences encoding HLLRCR-1 and either CD64 or CD64-GFP. CD64 andCD64-GFP are expressed on the surface of transfected cells and bind toconserved regions of human immunoglobulin G (IgG). Transfected cells areefficiently separated from nontransfected cells using magnetic beadscoated with either human IgG or antibody against CD64 (DYNAL, LakeSuccess N.Y.). mRNA can be purified from the cells using methods wellknown by those of skill in the art. Expression of mRNA encoding HLLRCR-1polypeptides and other genes of interest can be analyzed by northernanalysis or microarray techniques.

Example 10 Method of Screening for Compounds that Interact with theHLLRCR-1 Polypeptide

The following assays are designed to identify compounds that bind to theHLLRCR-1 polypeptide, bind to other cellular proteins that interact withthe HLLRCR-1 polypeptide, and to compounds that interfere with theinteraction of the HLLRCR-1 polypeptide with other cellular proteins.

Such compounds can include, but are not limited to, other cellularproteins. Specifically, such compounds can include, but are not limitedto, peptides, such as, for example, soluble peptides, including, but notlimited to Ig-tailed fusion peptides, comprising extracellular portionsof HLLRCR-1 polypeptide transmembrane receptors, and members of randompeptide libraries (see, e.g., Lam, K. S. et al., 1991, Nature 354:82-84;Houghton, R. et al., 1991, Nature 354:84-86), made of D-and/orL-configuration amino acids, phosphopeptides (including, but not limitedto, members of random or partially degenerate phosphopeptide libraries;see, e.g., Songyang, Z., et al., 1993, Cell 72:767-778), antibodies(including, but not limited to, polyclonal, monoclonal, humanized,anti-idiotypic, chimeric or single chain antibodies, and FAb,F(ab′).sub.2 and FAb expression libary fragments, and epitope-bindingfragments thereof), and small organic or inorganic molecules.

Compounds identified via assays such as those described herein can beuseful, for example, in elaborating the biological function of theHLLRCR-1 polypeptide, and for ameliorating symptoms of tumorprogression, for example. In instances, for example, whereby a tumorprogression state or disorder results from a lower overall level ofHLLRCR-1 expression, HLLRCR-1 polypeptide, and/or HLLRCR-1 polypeptideactivity in a cell involved in the tumor progression state or disorder,compounds that interact with the HLLRCR-1 polypeptide can include oneswhich accentuate or amplify the activity of the bound HLLRCR-1polypeptide. Such compounds would bring about an effective increase inthe level of HLLRCR-1 polypeptide activity, thus ameliorating symptomsof the tumor progression disorder or state. In instances wherebymutations within the HLLRCR-1 polypeptide cause aberrant HLLRCR-1polypeptides to be made which have a deleterious effect that leads totumor progression, compounds that bind HLLRCR-1 polypeptide can beidentified that inhibit the activity of the bound HLLRCR-1 polypeptide.Assays for testing the effectiveness of such compounds are known in theart and discussed, elsewhere herein.

Example 11 Method of Screening, In Vitro, Compounds that Bind to theHLLRCR-1 Polypeptide

In vitro systems can be designed to identify compounds capable ofbinding the HLLRCR-1 polypeptide of the invention. Compounds identifiedcan be useful, for example, in modulating the activity of wild typeand/or mutant HLLRCR-1 polypeptide, preferably mutant HLLRCR-1polypeptide, can be useful in elaborating the biological function of theHLLRCR-1 polypeptide, can be utilized in screens for identifyingcompounds that disrupt normal HLLRCR-1 polypeptide interactions, or canin themselves disrupt such interactions.

The principle of the assays used to identify compounds that bind to theHLLRCR-1 polypeptide involves preparing a reaction mixture of theHLLRCR-1 polypeptide and the test compound under conditions and for atime sufficient to allow the two components to interact and bind, thusforming a complex which can be removed and/or detected in the reactionmixture. These assays can be conducted in a variety of ways. Forexample, one method to conduct such an assay would involve anchoringHLLRCR-1 polypeptide or the test substance onto a solid phase anddetecting HLLRCR-1 polypeptide /test compound complexes anchored on thesolid phase at the end of the reaction. In one embodiment of such amethod, the HLLRCR-1 polypeptide can be anchored onto a solid surface,and the test compound, which is not anchored, can be labeled, eitherdirectly or indirectly.

In practice, microtitre plates can conveniently be utilized as the solidphase. The anchored component can be immobilized by non-covalent orcovalent attachments. Non-covalent attachment can be accomplished bysimply coating the solid surface with a solution of the protein anddrying. Alternatively, an immobilized antibody, preferably a monoclonalantibody, specific for the protein to be immobilized can be used toanchor the protein to the solid surface. The surfaces can be prepared inadvance and stored.

In order to conduct the assay, the nonimmobilized component is added tothe coated surface containing the anchored component. After the reactionis complete, unreacted components are removed (e.g., by washing) underconditions such that any complexes formed will remain immobilized on thesolid surface. The detection of complexes anchored on the solid surfacecan be accomplished in a number of ways. Where the previouslyimmobilized component is pre-labeled, the detection of label immobilizedon the surface indicates that complexes were formed. Where thepreviously nonimmobilized component is not pre-labeled, an indirectlabel can be used to detect complexes anchored on the surface; e.g.,using a labeled antibody specific for the immobilized component (theantibody, in turn, can be directly labeled or indirectly labeled with alabeled anti-Ig antibody).

Alternatively, a reaction can be conducted in a liquid phase, thereaction products separated from unreacted components, and complexesdetected; e.g., using an immobilized antibody specific for HLLRCR-1polypeptide or the test compound to anchor any complexes formed insolution, and a labeled antibody specific for the other component of thepossible complex to detect anchored complexes.

Example 12 Method for Identifying a Putative Ligand for the HLLRCR-1Polypeptide

Ligand binding assays provide a direct method for ascertaining receptorpharmacology and are adaptable to a high throughput format. A panel ofknown leucine-rich repeat containing protein purified ligands may beradiolabeled to high specific activity (50-2000 Ci/mmol) for bindingstudies. A determination is then made that the process of radiolabelingdoes not diminish the activity of the ligand towards its receptor. Assayconditions for buffers, ions, pH and other modulators such asnucleotides are optimized to establish a workable signal to noise ratiofor both membrane and whole cell receptor sources. For these assays,specific receptor binding is defined as total associated radioactivityminus the radioactivity measured in the presence of an excess ofunlabeled competing ligand. Where possible, more than one competingligand is used to define residual nonspecific binding.

A number of leucine-rich repeat containing protein ligands are known inthe art and are encompassed by the present invention.

Alternatively, the HLLRCR-1 polypeptide of the present invention mayalso be functionally screened against tissue extracts to identifynatural ligands. Extracts that produce positive functional responses canbe sequencially subfractionated until an activating ligand is isolatedidentified using methods well known in the art, some of which aredescribed herein.

Example 13 Method of Identifying Compounds that Interfere with HLLRCR-1Polypeptide/Cellular Product Interaction

The HLLRCR-1 polypeptide of the invention can, in vivo, interact withone or more cellular or extracellular macromolecules, such as proteins.Such macromolecules include, but are not limited to, polypeptides, andthose products identified via screening methods described, elsewhereherein. For the purposes of this discussion, such cellular andextracellular macromolecules are referred to herein as “bindingpartner(s)”. For the purpose of the present invention, “binding partner”may also encompass polypeptides, small molecule compounds,polysaccarides, lipids, and any other molecule or molecule typereferenced herein. Compounds that disrupt such interactions can beuseful in regulating the activity of the HLLRCR-1 polypeptide,especially mutant HLLRCR-1 polypeptide. Such compounds can include, butare not limited to molecules such as antibodies, peptides, and the likedescribed in elsewhere herein.

The basic principle of the assay systems used to identify compounds thatinterfere with the interaction between the HLLRCR-1 polypeptide and itscellular or extracellular binding partner or partners involves preparinga reaction mixture containing the HLLRCR-1 polypeptide, and the bindingpartner under conditions and for a time sufficient to allow the twoproducts to interact and bind, thus forming a complex. In order to testa compound for inhibitory activity, the reaction mixture is prepared inthe presence and absence of the test compound. The test compound can beinitially included in the reaction mixture, or can be added at a timesubsequent to the addition of HLLRCR-1 polypeptide and its cellular orextracellular binding partner. Control reaction mixtures are incubatedwithout the test compound or with a placebo. The formation of anycomplexes between the HLLRCR-1 polypeptide and the cellular orextracellular binding partner is then detected. The formation of acomplex in the control reaction, but not in the reaction mixturecontaining the test compound, indicates that the compound interfereswith the interaction of the HLLRCR-1 polypeptide and the interactivebinding partner. Additionally, complex formation within reactionmixtures containing the test compound and normal HLLRCR-1 polypeptidecan also be compared to complex formation within reaction mixturescontaining the test compound and mutant HLLRCR-1 polypeptide. Thiscomparison can be important in those cases wherein it is desirable toidentify compounds that disrupt interactions of mutant but not normalHLLRCR-1 polypeptide.

The assay for compounds that interfere with the interaction of theHLLRCR-1 polypeptide and binding partners can be conducted in aheterogeneous or homogeneous format. Heterogeneous assays involveanchoring either the HLLRCR-1 polypeptide or the binding partner onto asolid phase and detecting complexes anchored on the solid phase at theend of the reaction. In homogeneous assays, the entire reaction iscarried out in a liquid phase. In either approach, the order of additionof reactants can be varied to obtain different information about thecompounds being tested. For example, test compounds that interfere withthe interaction between the HLLRCR-1 polypeptide and the bindingpartners, e.g., by competition, can be identified by conducting thereaction in the presence of the test substance; i.e., by adding the testsubstance to the reaction mixture prior to or simultaneously with theHLLRCR-1 polypeptide and interactive cellular or extracellular bindingpartner. Alternatively, test compounds that disrupt preformed complexes,e.g. compounds with higher binding constants that displace one of thecomponents from the complex, can be tested by adding the test compoundto the reaction mixture after complexes have been formed. The variousformats are described briefly below.

In a heterogeneous assay system, either the HLLRCR-1 polypeptide or theinteractive cellular or extracellular binding partner, is anchored ontoa solid surface, while the non-anchored species is labeled, eitherdirectly or indirectly. In practice, microtitre plates are convenientlyutilized. The anchored species can be immobilized by non-covalent orcovalent attachments. Non-covalent attachment can be accomplished simplyby coating the solid surface with a solution of the HLLRCR-1 polypeptideor binding partner and drying. Alternatively, an immobilized antibodyspecific for the species to be anchored can be used to anchor thespecies to the solid surface. The surfaces can be prepared in advanceand stored.

In order to conduct the assay, the partner of the immobilized species isexposed to the coated surface with or without the test compound. Afterthe reaction is complete, unreacted components are removed (e.g., bywashing) and any complexes formed will remain immobilized on the solidsurface. The detection of complexes anchored on the solid surface can beaccomplished in a number of ways. Where the non-immobilized species ispre-labeled, the detection of label immobilized on the surface indicatesthat complexes were formed. Where the non-immobilized species is notpre-labeled, an indirect label can be used to detect complexes anchoredon the surface; e.g., using a labeled antibody specific for theinitially non-immobilized species (the antibody, in turn, can bedirectly labeled or indirectly labeled with a labeled anti-Ig antibody).Depending upon the order of addition of reaction components, testcompounds which inhibit complex formation or which disrupt preformedcomplexes can be detected.

Alternatively, the reaction can be conducted in a liquid phase in thepresence or absence of the test compound, the reaction productsseparated from unreacted components, and complexes detected; e.g., usingan immobilized antibody specific for one of the binding components toanchor any complexes formed in solution, and a labeled antibody specificfor the other partner to detect anchored complexes. Again, dependingupon the order of addition of reactants to the liquid phase, testcompounds which inhibit complex or which disrupt preformed complexes canbe identified.

In an alternate embodiment of the invention, a homogeneous assay can beused. In this approach, a preformed complex of the HLLRCR-1 polypeptideand the interactive cellular or extracellular binding partner product isprepared in which either the HLLRCR-1 polypeptide or their bindingpartners are labeled, but the signal generated by the label is quencheddue to complex formation (see, e.g., U.S. Pat. No. 4,109,496 byRubenstein which utilizes this approach for immunoassays).

The addition of a test substance that competes with and displaces one ofthe species from the preformed complex will result in the generation ofa signal above background. In this way, test substances which disruptHLLRCR-1 polypeptide—cellular or extracellular binding partnerinteraction can be identified.

In a particular embodiment, the HLLRCR-1 polypeptide can be prepared forimmobilization using recombinant DNA techniques known in the art. Forexample, the HLLRCR-1 polypeptide coding region can be fused to aglutathione-S-transferase (GST) gene using a fusion vector such aspGEX-5X-1, in such a manner that its binding activity is maintained inthe resulting fusion product. The interactive cellular or extracellularproduct can be purified and used to. raise a monoclonal antibody, usingmethods routinely practiced in the art and described above. Thisantibody can be labeled with the radioactive isotope sup.125 I, forexample, by methods routinely practiced in the art. In a heterogeneousassay, e.g., the GST-HLLRCR-1 polypeptide fusion product can be anchoredto glutathione-agarose beads. The interactive cellular or extracellularbinding partner product can then be added in the presence or absence ofthe test compound in a manner that allows interaction and binding tooccur. At the end of the reaction period, unbound material can be washedaway, and the labeled monoclonal antibody can be added to the system andallowed to bind to the complexed components. The interaction between theHLLRCR-1 polypeptide and the interactive cellular or extracellularbinding partner can be detected by measuring the amount of radioactivitythat remains associated with the glutathione-agarose beads. A successfulinhibition of the interaction by the test compound will result in adecrease in measured radioactivity.

Alternatively, the GST-HLLRCR-1 polypeptide fusion product and theinteractive cellular or extracellular binding partner product can bemixed together in liquid in the absence of the solid glutathione-agarosebeads. The test compound can be added either during or after the bindingpartners are allowed to interact. This mixture can then be added to theglutathione-agarose beads and unbound material is washed away. Again theextent of inhibition of the binding partner interaction can be detectedby adding the labeled antibody and measuring the radioactivityassociated with the beads.

In another embodiment of the invention, these same techniques can beemployed using peptide fragments that correspond to the binding domainsof the HLLRCR-1 polypeptide product and the interactive cellular orextracellular binding partner (in case where the binding partner is aproduct), in place of one or both of the full length products.

Any number of methods routinely practiced in the art can be used toidentify and isolate the protein's binding site. These methods include,but are not limited to, mutagenesis of one of the genes encoding one ofthe products and screening for disruption of binding in aco-immunoprecipitation assay. Compensating mutations in the geneencoding the second species in the complex can be selected. Sequenceanalysis of the genes encoding the respective products will reveal themutations that correspond to the region of the product involved ininteractive binding. Alternatively, one product can be anchored to asolid surface using methods described in this Section above, and allowedto interact with and bind to its labeled binding partner, which has beentreated with a proteolytic enzyme, such as trypsin. After washing, ashort, labeled peptide comprising the binding domain can remainassociated with the solid material, which can be isolated and identifiedby amino acid sequencing. Also, once the gene coding for the cellular orextracellular binding partner product is obtained, short gene segmentscan be engineered to express peptide fragments of the product, which canthen be tested for binding activity and purified or synthesized.

Example 14 Isolation of a Specific Clone from the Deposited Sample

The deposited material in the sample assigned the ATCC Deposit Numbercited in Table I for any given cDNA clone also may contain one or moreadditional plasmids, each comprising a cDNA clone different from thatgiven clone. Thus, deposits sharing the same ATCC Deposit Number containat least a plasmid for each cDNA clone identified in Table I. Typically,each ATCC deposit sample cited in Table I comprises a mixture ofapproximately equal amounts (by weight) of about 1-10 plasmid DNAs, eachcontaining a different cDNA clone and/or partial cDNA clone; but such adeposit sample may include plasmids for more or less than 2 cDNA clones.

Two approaches can be used to isolate a particular clone from thedeposited sample of plasmid DNA(s) cited for that clone in Table I.First, a plasmid is directly isolated by screening the clones using apolynucleotide probe corresponding to SEQ ID NO:1 and/or 3 and/or SEQ IDNO:3.

Particularly, a specific polynucleotide with 30-40 nucleotides issynthesized using an Applied Biosystems DNA synthesizer according to thesequence reported. The oligonucleotide is labeled, for instance, with32P-(-ATP using T4 polynucleotide kinase and purified according toroutine methods. (E.g., Maniatis et al., Molecular Cloning: A LaboratoryManual, Cold Spring Harbor Press, Cold Spring, N.Y. (1982).) The plasmidmixture is transformed into a suitable host, as indicated above (such asXL-1 Blue (Stratagene)) using techniques known to those of skill in theart, such as those provided by the vector supplier or in relatedpublications or patents cited above. The transformants are plated on1.5% agar plates (containing the appropriate selection agent, e.g.,ampicillin) to a density of about 150 transformants (colonies) perplate. These plates are screened using Nylon membranes according toroutine methods for bacterial colony screening (e.g., Sambrook et al.,Molecular Cloning: A Laboratory Manual, 2nd Edit., (1989), Cold SpringHarbor Laboratory Press, pages 1.93 to 1.104), or other techniques knownto those of skill in the art.

Alternatively, two primers of 17-20 nucleotides derived from both endsof the SEQ ID NO:1 and/or 3 and/or SEQ ID NO:3 (i.e., within the regionof SEQ ID NO:1 and/or 3 bounded by the 5′ NT and the 3′ NT of the clonedefined in Table I, and/or within the region of SEQ ID NO:1 and/or 3bounded by the 5′ NT and the 3′ NT of the clone defined in Table I) aresynthesized and used to amplify the desired cDNA using the depositedcDNA plasmid as a template. The polymerase chain reaction is carried outunder routine conditions, for instance, in 25 ul of reaction mixturewith 0.5 ug of the above cDNA template. A convenient reaction mixture is1.5-5 mM MgCl2, 0.01% (w/v) gelatin, 20 uM each of DATP, dCTP, dGTP,dTTP, 25 pmol of each primer and 0.25 Unit of Taq polymerase. Thirtyfive cycles of PCR (denaturation at 94 degree C. for 1 min; annealing at55 degree C. for 1 min; elongation at 72 degree C. for 1 min) areperformed with a Perkin-Elmer Cetus automated thermal cycler. Theamplified product is analyzed by agarose gel electrophoresis and the DNAband with expected molecular weight is excised and purified. The PCRproduct is verified to be the selected sequence by subcloning andsequencing the DNA product.

The polynucleotide(s) of the present invention, the polynucleotideencoding the polypeptide of the present invention, or the polypeptideencoded by the deposited clone may represent partial, or incompleteversions of the complete coding region (i.e., full-length gene). Severalmethods are known in the art for the identification of the 5′ or 3′non-coding and/or coding portions of a gene which may not be present inthe deposited clone. The methods that follow are exemplary and shouldnot be construed as limiting the scope of the invention. These methodsinclude but are not limited to, filter probing, clone enrichment usingspecific probes, and protocols similar or identical to 5′ and 3′ “RACE”protocols that are well known in the art. For instance, a method similarto 5′ RACE is available for generating the missing 5′ end of a desiredfull-length transcript. (Fromont-Racine et al., Nucleic Acids Res.21(7):1683-1684 (1993)).

Briefly, a specific RNA oligonucleotide is ligated to the 5′ ends of apopulation of RNA presumably containing full-length gene RNAtranscripts. A primer set containing a primer specific to the ligatedRNA oligonucleotide and a primer specific to a known sequence of thegene of interest is used to PCR amplify the 5′ portion of the desiredfull-length gene. This amplified product may then be sequenced and usedto generate the full-length gene.

This above method starts with total RNA isolated from the desiredsource, although poly-A+RNA can be used. The RNA preparation can then betreated with phosphatase if necessary to eliminate 5′ phosphate groupson degraded or damaged RNA that may interfere with the later RNA ligasestep. The phosphatase should then be inactivated and the RNA treatedwith tobacco acid pyrophosphatase in order to remove the cap structurepresent at the 5′ ends of messenger RNAs. This reaction leaves a 5′phosphate group at the 5′ end of the cap cleaved RNA which can then beligated to an RNA oligonucleotide using T4 RNA ligase.

This modified RNA preparation is used as a template for first strandcDNA synthesis using a gene specific oligonucleotide. The first strandsynthesis reaction is used as a template for PCR amplification of thedesired 5′ end using a primer specific to the ligated RNAoligonucleotide and a primer specific to the known sequence of the geneof interest. The resultant product is then sequenced and analyzed toconfirm that the 5′ end sequence belongs to the desired gene. Moreover,it may be advantageous to optimize the RACE protocol to increase theprobability of isolating additional 5′ or 3′ coding or non-codingsequences. Various methods of optimizing a RACE protocol are known inthe art, though a detailed description summarizing these methods can befound in B. C. Schaefer, Anal. Biochem., 227:255-273, (1995).

An alternative method for carrying out 5′ or 3′ RACE for theidentification of coding or non-coding sequences is provided by Frohman,M. A., et al., Proc. Nat'l. Acad. Sci. USA, 85:8998-9002 (1988).Briefly, a cDNA clone missing either the 5′ or 3′ end can bereconstructed to include the absent base pairs extending to thetranslational start or stop codon, respectively. In some cases, cDNAsare missing the start of translation, therefor. The following brieflydescribes a modification of this original 5′ RACE procedure. Poly A+ ortotal RNAs reverse transcribed with Superscript II (Gibco/BRL) and anantisense or I complementary primer specific to the cDNA sequence. Theprimer is removed from the reaction with a Microcon Concentrator(Amicon). The first-strand cDNA is then tailed with DATP and terminaldeoxynucleotide transferase (Gibco/BRL). Thus, an anchor sequence isproduced which is needed for PCR amplification. The second strand issynthesized from the dA-tail in PCR buffer, Taq DNA polymerase(Perkin-Elmer Cetus), an oligo-dT primer containing three adjacentrestriction sites (XhoIJ Sail and ClaI) at the 5′ end and a primercontaining just these restriction sites. This double-stranded cDNA isPCR amplified for 40 cycles with the same primers as well as a nestedcDNA-specific antisense primer. The PCR products are size-separated onan ethidium bromide-agarose gel and the region of gel containing cDNAproducts the predicted size of missing protein-coding DNA is removed.cDNA is purified from the agarose with the Magic PCR Prep kit (Promega),restriction digested with XhoI or SalI, and ligated to a plasmid such aspBluescript SKII (Stratagene) at XhoI and EcoRV sites. This DNA istransformed into bacteria and the plasmid clones sequenced to identifythe correct protein-coding inserts. Correct 5′ ends are confirmed bycomparing this sequence with the putatively identified homologue andoverlap with the partial cDNA clone. Similar methods known in the artand/or commercial kits are used to amplify and recover 3′ ends.

Several quality-controlled kits are commercially available for purchase.Similar reagents and methods to those above are supplied in kit formfrom Gibco/BRL for both 5′ and 3′ RACE for recovery of full lengthgenes. A second kit is available from Clontech which is a modificationof a related technique, SLIC (single-stranded ligation tosingle-stranded cDNA), developed by Dumas et al., Nucleic Acids Res.,19:5227-32(1991). The major differences in procedure are that the RNA isalkaline hydrolyzed after reverse transcription and RNA ligase is usedto join a restriction site-containing anchor primer to the first-strandcDNA. This obviates the necessity for the dA-tailing reaction whichresults in a polyT stretch that is difficult to sequence past.

An alternative to generating 5′ or 3′ cDNA from RNA is to use cDNAlibrary double- stranded DNA. An asymmetric PCR-amplified antisense cDNAstrand is synthesized with an antisense cDNA-specific primer and aplasmid-anchored primer. These primers are removed and a symmetric PCRreaction is performed with a nested cDNA-specific antisense primer andthe plasmid-anchored primer.

RNA Ligase Protocol for Generating the 5′ or 3′ end Sequences to ObtainFull Length Genes

Once a gene of interest is identified, several methods are available forthe identification of the 5′ or 3′ portions of the gene which may not bepresent in the original cDNA plasmid. These methods include, but are notlimited to, filter probing, clone enrichment using specific probes andprotocols similar and identical to 5′ and 3′RACE. While the full-lengthgene may be present in the library and can be identified by probing, auseful method for generating the 5′ or 3′ end is to use the existingsequence information from the original cDNA to generate the missinginformation. A method similar to 5′RACE is available for generating themissing 5′ end of a desired full-length gene. (This method was publishedby Fromont-Racine et al., Nucleic Acids Res., 21(7): 1683-1684 (1993)).Briefly, a specific RNA oligonucleotide is ligated to the 5′ ends of apopulation of RNA presumably 30 containing full-length gene RNAtranscript and a primer set containing a primer specific to the ligatedRNA oligonucleotide and a primer specific to a known sequence of thegene of interest, is used to PCR amplify the 5′ portion of the desiredfull length gene which may then be sequenced and used to generate thefull length gene. This method starts with total RNA isolated from thedesired source, poly A RNA may be used but is not a prerequisite forthis procedure. The RNA preparation may then be treated with phosphataseif necessary to eliminate 5′ phosphate groups on degraded or damaged RNAwhich may interfere with the later RNA ligase step. The phosphatase ifused is then inactivated and the RNA is treated with tobacco acidpyrophosphatase in order to remove the cap structure present at the 5′ends of messenger RNAs. This reaction leaves a 5′ phosphate group at the5′ end of the cap cleaved RNA which can then be ligated to an RNAoligonucleotide using T4 RNA ligase. This modified RNA preparation canthen be used as a template for first strand cDNA synthesis using a genespecific oligonucleotide. The first strand synthesis reaction can thenbe used as a template for PCR amplification of the desired 5′ end usinga primer specific to the ligated RNA oligonucleotide and a primerspecific to the known sequence of the apoptosis related of interest. Theresultant product is then sequenced and analyzed to confirm that the 5′end sequence belongs to the relevant apoptosis related.

Example 15 Bacterial Expression of a HLLRCR-1 Polypeptide

A polynucleotide encoding a polypeptide of the present invention isamplified using PCR oligonucleotide primers corresponding to the 5′ and3′ ends of the DNA sequence, as outlined in Example 13, to synthesizeinsertion fragments. The primers used to amplify the cDNA insert shouldpreferably contain restriction sites, such as BamHI and XbaI, at the 5′end of the primers in order to clone the amplified product into theexpression vector. For example, BamHI and XbaI correspond to therestriction enzyme sites on the bacterial expression vector pQE-9.(Qiagen, Inc., Chatsworth, Calif.). This plasmid vector encodesantibiotic resistance (Ampr), a bacterial origin of replication (ori),an IPTG-regulatable promoter/operator (P/O), a ribosome binding site(RBS), a 6-histidine tag (6-His), and restriction enzyme cloning sites.

The pQE-9 vector is digested with BamHI and XbaI and the amplifiedfragment is ligated into the pQE-9 vector maintaining the reading frameinitiated at the bacterial RBS. The ligation mixture is then used totransform the E. coli strain M15/rep4 (Qiagen, Inc.) which containsmultiple copies of the plasmid pREP4, that expresses the lacI repressorand also confers kanamycin resistance (Kanr). Transformants areidentified by their ability to grow on LB plates andampicillin/kanamycin resistant colonies are selected. Plasmid DNA isisolated and confirmed by restriction analysis.

Clones containing the desired constructs are grown overnight (O/N) inliquid culture in LB media supplemented with both Amp (100 ug/ml) andKan (25 ug/ml). The O/N culture is used to inoculate a large culture ata ratio of 1:100 to 1:250. The cells are grown to an optical density 600(O.D.600) of between 0.4 and 0.6. IPTG (Isopropyl-B-D-thiogalactopyranoside) is then added to a final concentration of 1 mM. IPTG inducesby inactivating the lacI repressor, clearing the P/O leading toincreased gene expression.

Cells are grown for an extra 3 to 4 hours. Cells are then harvested bycentrifugation (20 mins at 6000×g). The cell pellet is solubilized inthe chaotropic agent 6 Molar Guanidine HCl by stirring for 3-4 hours at4 degree C. The cell debris is removed by centrifugation, and thesupernatant containing the polypeptide is loaded onto anickel-nitrilo-tri-acetic acid (“Ni—NTA”) affinity resin column(available from QIAGEN, Inc., supra). Proteins with a 6× His tag bind tothe Ni—NTA resin with high affinity and can be purified in a simpleone-step procedure (for details see: The QIAexpressionist (1995) QIAGEN,Inc., supra).

Briefly, the supernatant is loaded onto the column in 6 M guanidine-HCl,pH 8, the column is first washed with 10 volumes of 6 M guanidine-HCl,pH 8, then washed with 10 volumes of 6 M guanidine-HCl pH 6, and finallythe polypeptide is eluted with 6 M guanidine-HCl, pH 5.

The purified protein is then renatured by dialyzing it againstphosphate-buffered saline (PBS) or 50 mM Na-acetate, pH 6 buffer plus200 mM NaCl. Alternatively, the protein can be successfully refoldedwhile immobilized on the Ni—NTA column. The recommended conditions areas follows: renature using a linear 6M-1M urea gradient in 500 mM NaCl,20% glycerol, 20 mM Tris/HCl pH 7.4, containing protease inhibitors. Therenaturation should be performed over a period of 1.5 hours or more.After renaturation the proteins are eluted by the addition of 250 mMimidazole. Imidazole is removed by a final dialyzing step against PBS or50 mM sodium acetate pH 6 buffer plus 200 mM NaCl. The purified proteinis stored at 4 degree C or frozen at −80 degree C.

Example 16 Purification of a HLLRCR-1 Polypeptide from an Inclusion Body

The following alternative method can be used to purify a polypeptideexpressed in E coli when it is present in the form of inclusion bodies.Unless otherwise specified, all of the following steps are conducted at4-10 degree C.

Upon completion of the production phase of the E. coli fermentation, thecell culture is cooled to 4-10 degree C. and the cells harvested bycontinuous centrifugation at 15,000 rpm (Heraeus Sepatech). On the basisof the expected yield of protein per unit weight of cell paste and theamount of purified protein required, an appropriate amount of cellpaste, by weight, is suspended in a buffer solution containing 100 mMTris, 50 mM EDTA, pH 7.4. The cells are dispersed to a homogeneoussuspension using a high shear mixer.

The cells are then lysed by passing the solution through amicrofluidizer (Microfluidics, Corp. or APV Gaulin, Inc.) twice at4000-6000 psi. The homogenate is then mixed with NaCl solution to afinal concentration of 0.5 M NaCl, followed by centrifugation at 7000×gfor 15 min. The resultant pellet is washed again using 0.5M, NaCi, 100mM Tris, 50 mM EDTA, pH 7.4.

The resulting washed inclusion bodies are solubilized with 1.5 Mguanidine hydrochloride (GuHCl) for 2-4 hours. After 7000×gcentrifugation for 15 min., the pellet is discarded and the polypeptidecontaining supernatant is incubated at 4 degree C. overnight to allowfurther GuHCI extraction.

Following high speed centrifugation (30,000×g) to remove insolubleparticles, the GuHCl solubilized protein is refolded by quickly mixingthe GuHCl extract with 20 volumes of buffer containing 50 mM sodium, pH4.5, 150 mM NaCl, 2 mM EDTA by vigorous stirring. The refolded dilutedprotein solution is kept at 4 degree C. without mixing for 12 hoursprior to further purification steps.

To clarify the refolded polypeptide solution, a previously preparedtangential filtration unit equipped with 0.16 um membrane filter withappropriate surface area (e.g., Filtron), equilibrated with 40 mM sodiumacetate, pH 6.0 is employed. The filtered sample is loaded onto a cationexchange resin (e.g., Poros HS-50, Perceptive Biosystems). The column iswashed with 40 mM sodium acetate, pH 6.0 and eluted with 250 mM, 500 mM,1000 mM, and 1500 mM NaCl in the same buffer, in a stepwise manner. Theabsorbance at 280 nm of the effluent is continuously monitored.Fractions are collected and further analyzed by SDS-PAGE.

Fractions containing the polypeptide are then pooled and mixed with 4volumes of water. The diluted sample is then loaded onto a previouslyprepared set of tandem columns of strong anion (Poros HQ-50, PerceptiveBiosystems) and weak anion (Poros CM-20, Perceptive Biosystems) exchangeresins. The columns are equilibrated with 40 mM sodium acetate, pH 6.0.Both columns are washed with 40 mM sodium acetate, pH 6.0, 200 mM NaCl.The CM-20 column is then eluted using a 10 column volume linear gradientranging from 0.2 M NaCl, 50 mM sodium acetate, pH 6.0 to 1.0 M NaCl, 50mM sodium acetate, pH 6.5. Fractions are collected under constant A280monitoring of the effluent. Fractions containing the polypeptide(determined, for instance, by 16% SDS-PAGE) are then pooled.

The resultant polypeptide should exhibit greater than 95% purity afterthe above refolding and purification steps. No major contaminant bandsshould be observed from Coomassie blue stained 16% SDS-PAGE gel when 5ug of purified protein is loaded. The purified protein can also betested for endotoxin/LPS contamination, and typically the LPS content isless than 0.1 ng/ml according to LAL assays.

Example 17 Cloning and Expression of a HLLRCR-1 Polypeptide in aBaculovirus Expression System

In this example, the plasmid shuttle vector pAc373 is used to insert apolynucleotide into a baculovirus to express a polypeptide. A typicalbaculovirus expression vector contains the strong polyhedrin promoter ofthe Autographa californica nuclear polyhedrosis virus (AcMNPV) followedby convenient restriction sites, which may include, for example BamHI,Xba I and Asp718. The polyadenylation site of the simian virus 40(“SV40”) is often used for efficient polyadenylation. For easy selectionof recombinant virus, the plasmid contains the beta-galactosidase genefrom E. coli under control of a weak Drosophila promoter in the sameorientation, followed by the polyadenylation signal of the polyhedringene. The inserted genes are flanked on both sides by viral sequencesfor cell-mediated homologous recombination with wild-type viral DNA togenerate a viable virus that express the cloned polynucleotide.

Many other baculovirus vectors can be used in place of the vector above,such as pVL941 and pAcIM1, as one skilled in the art would readilyappreciate, as long as the construct provides appropriately locatedsignals for transcription, translation, secretion and the like,including a signal peptide and an in-frame AUG as required. Such vectorsare described, for instance, in Luckow et al., Virology 170:31-39(1989).

A polynucleotide encoding a polypeptide of the present invention isamplified using PCR oligonucleotide primers corresponding to the 5′ and3′ ends of the DNA sequence, as outlined in Example 13, to synthesizeinsertion fragments. The primers used to amplify the cDNA insert shouldpreferably contain restriction sites at the 5′ end of the primers inorder to clone the amplified product into the expression vector.Specifically, the cDNA sequence contained in the deposited clone,including the AUG initiation codon and the naturally associated leadersequence identified elsewhere herein (if applicable), is amplified usingthe PCR protocol described in Example 13. If the naturally occurringsignal sequence is used to produce the protein, the vector used does notneed a second signal peptide. Alternatively, the vector can be modifiedto include a baculovirus leader sequence, using the standard methodsdescribed in Summers et al., “A Manual of Methods for BaculovirusVectors and Insect Cell Culture Procedures,” Texas AgriculturalExperimental Station Bulletin No. 1555 (1987).

The amplified fragment is isolated from a 1% agarose gel using acommercially available kit (“Geneclean,” BIO 101 Inc., La Jolla,Calif.). The fragment then is digested with appropriate restrictionenzymes and again purified on a 1% agarose gel.

The plasmid is digested with the corresponding restriction enzymes andoptionally, can be dephosphorylated using calf intestinal phosphatase,using routine procedures known in the art. The DNA is then isolated froma 1% agarose gel using a commercially available kit (“Geneclean” BIO 101Inc., La Jolla, Calif.).

The fragment and the dephosphorylated plasmid are ligated together withT4 DNA ligase. E. coli HB101 or other suitable E. coli hosts such asXL-1 Blue (Stratagene Cloning Systems, La Jolla, Calif.) cells aretransformed with the ligation mixture and spread on culture plates.Bacteria containing the plasmid are identified by digesting DNA fromindividual colonies and analyzing the digestion product by gelelectrophoresis. The sequence of the cloned fragment is confirmed by DNAsequencing.

Five ug of a plasmid containing the polynucleotide is co-transformedwith 1.0 ug of a commercially available linearized baculovirus DNA(“BaculoGoldtm baculovirus DNA”, Pharmingen, San Diego, Calif.), usingthe lipofection method described by Felgner et al., Proc. Natl. Acad.Sci. USA 84:7413-7417 (1987). One ug of BaculoGoldtm virus DNA and 5 ugof the plasmid are mixed in a sterile well of a microtiter platecontaining 50 ul of serum-free Grace's medium (Life Technologies Inc.,Gaithersburg, Md.). Afterwards, 10 ul Lipofectin plus 90 ul Grace'smedium are added, mixed and incubated for 15 minutes at roomtemperature. Then the transfection mixture is added drop-wise to Sf9insect cells (ATCC CRL 1711) seeded in a 35 mm tissue culture plate with1 ml Grace's medium without serum. The plate is then incubated for 5hours at 27 degrees C. The transfection solution is then removed fromthe plate and 1 ml of Grace's insect medium supplemented with 10% fetalcalf serum is added. Cultivation is then continued at 27 degrees C. forfour days.

After four days the supernatant is collected and a plaque assay isperformed, as described by Summers and Smith, supra. An agarose gel with“Blue Gal” (Life Technologies Inc., Gaithersburg) is used to allow easyidentification and isolation of gal-expressing clones, which produceblue-stained plaques. (A detailed description of a “plaque assay” ofthis type can also be found in the user's guide for insect cell cultureand baculovirology distributed by Life Technologies Inc., Gaithersburg,page 9-10.) After appropriate incubation, blue stained plaques arepicked with the tip of a micropipettor (e.g., Eppendorf). The agarcontaining the recombinant viruses is then resuspended in amicrocentrifuge tube containing 200 ul of Grace's medium and thesuspension containing the recombinant baculovirus is used to infect Sf9cells seeded in 35 mm dishes. Four days later the supernatants of theseculture dishes are harvested and then they are stored at 4 degree C.

To verify the expression of the polypeptide, Sf9 cells are grown inGrace's medium supplemented with 10% heat-inactivated FBS. The cells areinfected with the recombinant baculovirus containing the polynucleotideat a multiplicity of infection (“MOI”) of about 2. If radiolabeledproteins are desired, 6 hours later the medium is removed and isreplaced with SF900 II medium minus methionine and cysteine (availablefrom Life Technologies Inc., Rockville, Md.). After 42 hours, 5 uCi of35S-methionine and 5 uCi 35S-cysteine (available from Amersham) areadded. The cells are further incubated for 16 hours and then areharvested by centrifugation. The proteins in the supernatant as well asthe intracellular proteins are analyzed by SDS-PAGE followed byautoradiography (if radiolabeled).

Microsequencing of the amino acid sequence of the amino terminus ofpurified protein may be used to determine the amino terminal sequence ofthe produced protein.

Example 18 Expression of a HLLRCR-1 Polypeptide in Mammalian Cells

The polypeptide of the present invention can be expressed in a mammaliancell. A typical mammalian expression vector contains a promoter element,which mediates the initiation of transcription of mRNA, a protein codingsequence, and signals required for the termination of transcription andpolyadenylation of the transcript. Additional elements includeenhancers, Kozak sequences and intervening sequences flanked by donorand acceptor sites for RNA splicing. Highly efficient transcription isachieved with the early and late promoters from SV40, the long terminalrepeats (LTRs) from Retroviruses, e.g., RSV, HTLVI, HIVI and the earlypromoter of the cytomegalovirus (CMV). However, cellular elements canalso be used (e.g., the human actin promoter).

Suitable expression vectors for use in practicing the present inventioninclude, for example, vectors such as pSVL and pMSG (Pharmacia, Uppsala,Sweden), pRSVcat (ATCC 37152), pSV2dhfr (ATCC 37146), pBC12MI (ATCC67109), pCMVSport 2.0, and pCMVSport 3.0. Mammalian host cells thatcould be used include, human Hela, 293, H9 and Jurkat cells, mouseNIH3T3 and C127 cells, Cos 1, Cos 7 and CVI, quail QC1-3 cells, mouse Lcells and Chinese hamster ovary (CHO) cells.

Alternatively, the polypeptide can be expressed in stable cell linescontaining the polynucleotide integrated into a chromosome. Theco-transformation with a selectable marker such as dhfr, gpt, neomycin,hygromycin allows the identification and isolation of the transformedcells.

The transformed gene can also be amplified to express large amounts ofthe encoded protein. The DHFR (dihydrofolate reductase) marker is usefulin developing cell lines that carry several hundred or even severalthousand copies of the gene of interest. (See, e.g., Alt, F. W., et al.,J. Biol. Chem. . . . 253:1357-1370 (1978); Hamlin, J. L. and Ma, C.,Biochem. et Biophys. Acta, 1097:107-143 (1990); Page, M. J. andSydenham, M. A., Biotechnology 9:64-68 (1991).) Another useful selectionmarker is the enzyme glutamine synthase (GS) (Murphy et al., Biochem J.227:277-279 (1991); Bebbington et al., Bio/Technology 10:169-175 (1992).Using these markers, the mammalian cells are grown in selective mediumand the cells with the highest resistance are selected. These cell linescontain the amplified gene(s) integrated into a chromosome. Chinesehamster ovary (CHO) and NSO cells are often used for the production ofproteins.

A polynucleotide of the present invention is amplified according to theprotocol outlined in herein. If the naturally occurring signal sequenceis used to produce the protein, the vector does not need a second signalpeptide. Alternatively, if the naturally occurring signal sequence isnot used, the vector can be modified to include a heterologous signalsequence. (See, e.g., WO 96/34891.) The amplified fragment is isolatedfrom a 1% agarose gel using a commercially available kit (“Geneclean,”BIO 101 Inc., La Jolla, Calif.). The fragment then is digested withappropriate restriction enzymes and again purified on a 1% agarose gel.

The amplified fragment is then digested with the same restriction enzymeand purified on a 1% agarose gel. The isolated fragment and thedephosphorylated vector are then ligated with T4 DNA ligase. E. coliHB101 or XL-1 Blue cells are then transformed and bacteria areidentified that contain the fragment inserted into plasmid pC6 using,for instance, restriction enzyme analysis.

Chinese hamster ovary cells lacking an active DHFR gene is used fortransformation. Five pg of an expression plasmid is cotransformed with0.5 ug of the plasmid pSVneo using lipofectin (Felgner et al., supra).The plasmid pSV2-neo contains a dominant selectable marker, the neo genefrom Tn5 encoding an enzyme that confers resistance to a group ofantibiotics including G418. The cells are seeded in alpha minus MEMsupplemented with 1 mg/ml G418. After 2 days, the cells are trypsinizedand seeded in hybridoma cloning plates (Greiner, Germany) in alpha minusMEM supplemented with 10, 25, or 50 ng/ml of methotrexate plus 1 mg/mlG418. After about 10-14 days single clones are trypsinized and thenseeded in 6-well petri dishes or 10 ml flasks using differentconcentrations of methotrexate (50 nM, 100 nM, 200 nM, 400 nM, 800 nM).Clones growing at the highest concentrations of methotrexate are thentransferred to new 6-well plates containing even higher concentrationsof methotrexate (1 uM, 2 uM, 5 uM, 10 mM, 20 mM). The same procedure isrepeated until clones are obtained which grow at a concentration of100-200 uM. Expression of the desired gene product is analyzed, forinstance, by SDS-PAGE and Western blot or by reversed phase HPLCanalysis.

Example 19 Protein Fusions of the HLLRCR-1 Polypeptide

The polypeptides of the present invention are preferably fused to otherproteins. These fusion proteins can be used for a variety ofapplications. For example, fusion of the present polypeptides toHis-tag, HA-tag, protein A, IgG domains, and maltose binding proteinfacilitates purification. (See Example described herein; see also EP A394,827; Traunecker, et al., Nature 331:84-86 (1988).) Similarly, fusionto IgG-1, IgG-3, and albumin increases the half-life time in vivo.Nuclear localization signals fused to the polypeptides of the presentinvention can target the protein to a specific subcellular localization,while covalent heterodimer or homodimers can increase or decrease theactivity of a fusion protein. Fusion proteins can also create chimericmolecules having more than one function. Finally, fusion proteins canincrease solubility and/or stability of the fused protein compared tothe non-fused protein. All of the types of fusion proteins describedabove can be made by modifying the following protocol, which outlinesthe fusion of a polypeptide to an IgG molecule.

Briefly, the human Fc portion of the IgG molecule can be PCR amplified,using primers that span the 5′ and 3′ ends of the sequence describedbelow. These primers also should have convenient restriction enzymesites that will facilitate cloning into an expression vector, preferablya mammalian expression vector. Note that the polynucleotide is clonedwithout a stop codon, otherwise a fusion protein will not be produced.

The naturally occurring signal sequence may be used to produce theprotein (if applicable). Alternatively, if the naturally occurringsignal sequence is not used, the vector can be modified to include aheterologous signal sequence. (See, e.g., WO 96/34891 and/or U.S. Pat.No. 6,066,781, supra.) (SEQ ID NO:52)GGGATCCGGAGCCCAAATCTTCTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAATTCGAGGGTGCACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACTCCTGAGGTCACATGCGTGGTGGTGGACGTAAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAACCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCAAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGAGTGCGACGGCCGCGACTCTAGAGGAT

Example 20 Regulation of Protein Expression Via Controlled Aggregationin the Endoplasmic Reticulum

As described more particularly herein, proteins regulate diversecellular processes in higher organisms, ranging from rapid metabolicchanges to growth and differentiation. Increased production of specificproteins could be used to prevent certain diseases and/or diseasestates. Thus, the ability to modulate the expression of specificproteins in an organism would provide significant benefits.

Numerous methods have been developed to date for introducing foreigngenes, either under the control of an inducible, constitutively active,or endogenous promoter, into organisms. Of particular interest are theinducible promoters (see, M. Gossen, et al., Proc. Natl. Acad. Sci.USA., 89:5547 (1992); Y. Wang, et al., Proc. Natl. Acad. Sci. USA,91:8180 (1994), D. No., et al., Proc. Natl. Acad. Sci. USA, 93:3346(1996); and V. M. Rivera, et al., Nature Med, 2:1028 (1996); in additionto additional examples disclosed elsewhere herein). In one example, thegene for erthropoietin (Epo) was transferred into mice and primatesunder the control of a small molecule inducer for expression (e.g.,tetracycline or rapamycin) (see, D. Bohl, et al., Blood, 92:1512,(1998); K. G. Rendahl, et al., Nat. Biotech, 16:757, (1998); V. M.Rivera, et al., Proc. Natl. Acad. Sci. USA, 96:8657 (1999); and X. Ye etal., Science, 283:88 (1999). Although such systems enable efficientinduction of the gene of interest in the organism upon addition of theinducing agent (i.e., tetracycline, rapamycin, etc,.), the levels ofexpression tend to peak at 24 hours and trail off to background levelsafter 4 to 14 days. Thus, controlled transient expression is virtuallyimpossible using these systems, though such control would be desirable.

A new alternative method of controlling gene expression levels of aprotein from a transgene (i.e., includes stable and transienttransformants) has recently been elucidated (V. M. Rivera., et al.,Science, 287:826-830, (2000)). This method does not control geneexpression at the level of the mRNA like the aforementioned systems.Rather, the system controls the level of protein in an active secretedform. In the absence of the inducing agent, the protein aggregates inthe ER and is not secreted. However, addition of the inducing agentresults in dis-aggregation of the protein and the subsequent secretionfrom the ER. Such a system affords low basal secretion, rapid, highlevel secretion in the presence of the inducing agent, and rapidcessation of secretion upon removal of the inducing agent. In fact,protein secretion reached a maximum level within 30 minutes ofinduction, and a rapid cessation of secretion within 1 hour of removingthe inducing agent. The method is also applicable for controlling thelevel of production for membrane proteins.

Detailed methods are presented in V. M. Rivera., et al., Science,287:826-830, (2000)), briefly:

Fusion protein constructs are created using polynucleotide sequences ofthe present invention with one or more copies (preferably at least 2, 3,4, or more) of a conditional aggregation domain (CAD) a domain thatinteracts with itself in a ligand-reversible manner (i.e., in thepresence of an inducing agent) using molecular biology methods known inthe art and discussed elsewhere herein. The CAD domain may be the mutantdomain isolated from the human FKBP12 (Phe³⁶ to Met) protein (asdisclosed in V. M. Rivera., et al., Science, 287:826-830, (2000), oralternatively other proteins having domains with similarligand-reversible, self-aggregation properties. As a principle of designthe fusion protein vector would contain a furin cleavage sequenceoperably linked between the polynucleotides of the present invention andthe CAD domains. Such a cleavage site would enable the proteolyticcleavage of the CAD domains from the polypeptide of the presentinvention subsequent to secretion from the ER and upon entry into thetrans-Golgi (J. B. Denault, et al., FEBS Lett., 379:113, (1996)).Alternatively, the skilled artisan would recognize that any proteolyticcleavage sequence could be substituted for the furin sequence providedthe substituted sequence is cleavable either endogenously (e.g., thefurin sequence) or exogenously (e.g., post secretion, post purification,post production, etc.). The preferred sequence of each feature of thefusion protein construct, from the 5′ to 3′ direction with each featurebeing operably linked to the other, would be a promoter, signalsequence, “X” number of (CAD)x domains, the furin sequence (or otherproteolytic sequence), and the coding sequence of the polypeptide of thepresent invention. The artisan would appreciate that the promotor andsignal sequence, independent from the other, could be either theendogenous promotor or signal sequence of a polypeptide of the presentinvention, or alternatively, could be a heterologous signal sequence andpromotor.

The specific methods described herein for controlling protein secretionlevels through controlled ER aggregation are not meant to be limitingare would be generally applicable to any of the polynucleotides andpolypeptides of the present invention, including variants, homologues,orthologs, and fragments therein.

Example 21 Alteration of Protein Glycosylation Sites to EnhanceCharacteristics of Polypeptides of the Invention

Many eukaryotic cell surface proteins are post-translationally processedto incorporate N-linked and O-linked carbohydrates (Komfeld and Komfeld(1985) Annu. Rev. Biochem. 54:631-64; Rademacher et al., (1988) Annu.Rev. Biochem. 57:785-838). Protein glycosylation is thought to serve avariety of functions including: augmentation of protein folding,inhibition of protein aggregation, regulation of intracellulartrafficking to organelles, increasing resistance to proteolysis,modulation of protein antigenicity, and mediation of intercellularadhesion (Fieldler and Simons (1995) Cell, 81:309-312; Helenius (1994)Mol. Biol. Of the Cell 5:253-265; Olden et al., (1978) Cell, 13:461-473;Caton et al., (1982) Cell, 37:417-427; Alexamnder and Elder (1984),Science, 226:1328-1330; and Flack et al., (1994), J. Biol. Chem.,269:14015-14020). In higher organisms, the nature and extent ofglycosylation can markedly affect the circulating half-life andbio-availability of proteins by mechanisms involving receptor mediateduptake and clearance (Ashwell and Morrell, (1974), Adv. Enzymol.,41:99-128; Ashwell and Harford (1982), Ann. Rev. Biochem., 51:531-54).Receptor systems have been identified that are thought to play a majorrole in the clearance of serum proteins through recognition of variouscarbohydrate structures on the glycoproteins (Stockert (1995), Physiol.Rev., 75:591-609; Kery et al., (1992), Arch. Biochem. Biophys.,298:49-55). Thus, production strategies resulting in incompleteattachment of terminal sialic acid residues might provide a means ofshortening the bioavailability and half-life of glycoproteins.Conversely, expression strategies resulting in saturation of terminalsialic acid attachment sites might lengthen protein bioavailability andhalf-life.

In the development of recombinant glycoproteins for use aspharmaceutical products, for example, it has been speculated that thepharmacodynamics of recombinant proteins can be modulated by theaddition or deletion of glycosylation sites from a glycoproteins primarystructure (Berman and Lasky (1985a) Trends in Biotechnol., 3:51-53).However, studies have reported that the deletion of N-linkedglycosylation sites often impairs intracellular transport and results inthe intracellular accumulation of glycosylation site variants (Machamerand Rose (1988), J. Biol Chem., 263:5955-5960; Gallagher et al., (1992),J. Virology., 66:7136-7145; Collier et al., (1993), Biochem.,32:7818-7823; Claffey et al., (1995) Biochemica et Biophysica Acta,1246:1-9; Dube et al., (1988), J. Biol. Chem. 263:17516-17521). Whileglycosylation site variants of proteins can be expressedintracellularly, it has proved difficult to recover useful quantitiesfrom growth conditioned cell culture medium.

Moreover, it is unclear to what extent a glycosylation site in onespecies will be recognized by another species glycosylation machinery.Due to the importance of glycosylation in protein metabolism,particularly the secretion and/or expression of the protein, whether aglycosylation signal is recognized may profoundly determine a proteinsability to be expressed, either endogenously or recombinately, inanother organism (i.e., expressing a human protein in E. coli, yeast, orviral organisms; or an E. coli, yeast, or viral protein in human, etc.).Thus, it may be desirable to add, delete, or modify a glycosylationsite, and possibly add a glycosylation site of one species to a proteinof another species to improve the proteins functional, bioprocesspurification, and/or structural characteristics (e.g., a polypeptide ofthe present invention).

A number of methods may be employed to identify the location ofglycosylation sites within a protein. One preferred method is to run thetranslated protein sequence through the PROSITE computer program (SwissInstitute of Bioinformatics), or alternatively, the MOTIF algorithm(Genetics Computer Group, Inc.). Once identified, the sites could besystematically deleted, or impaired, at the level of the DNA usingmutagenesis methodology known in the art and available to the skilledartisan, Preferably using PCR-directed mutagenesis (See Maniatis,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, ColdSpring, N.Y. (1982)). Similarly, glycosylation sites could be added, ormodified at the level of the DNA using similar methods, preferably PCRmethods (See, Maniatis, supra). The results of modifying theglycosylation sites for a particular protein (e.g., solubility,secretion potential, activity, aggregation, proteolytic resistance,etc.) could then be analyzed using methods know in the art.

The skilled artisan would acknowledge the existence of other computeralgorithms capable of predicting the location of glycosylation siteswithin a protein. For example, the Motif computer program (GeneticsComputer Group suite of programs) provides this function, as well.

Example 22 Method of Enhancing the Biological Activity/FunctionalCharacteristics of Invention Through Molecular Evolution

Although many of the most biologically active proteins known are highlyeffective for their specified function in an organism, they oftenpossess characteristics that make them undesirable for transgenic,therapeutic, and/or industrial applications. Among these traits, a shortphysiological half-life is the most prominent problem, and is presenteither at the level of the protein, or the level of the proteins mRNA.The ability to extend the half-life, for example, would be particularlyimportant for a proteins use in gene therapy, transgenic animalproduction, the bioprocess production and purification of the protein,and use of the protein as a chemical modulator among others. Therefore,there is a need to identify novel variants of isolated proteinspossessing characteristics which enhance their application as atherapeutic for treating diseases of animal origin, in addition to theproteins applicability to common industrial and pharmaceuticalapplications.

Thus, one aspect of the present invention relates to the ability toenhance specific characteristics of invention through directed molecularevolution. Such an enhancement may, in a non-limiting example, benefitthe inventions utility as an essential component in a kit, theinventions physical attributes such as its solubility, structure, orcodon optimization, the inventions specific biological activity,including any associated enzymatic activity, the proteins enzymekinetics, the proteins Ki, Kcat, Km, Vmax, Kd, protein-protein activity,protein-DNA binding activity, antagonist/inhibitory activity (includingdirect or indirect interaction), agonist activity (including direct orindirect interaction), the proteins antigenicity (e.g., where it wouldbe desirable to either increase or decrease the antigenic potential ofthe protein), the immunogenicity of the protein, the ability of theprotein to form dimers, trimers, or multimers with either itself orother proteins, the antigenic efficacy of the invention, including itssubsequent use a preventative treatment for disease or disease states,or as an effector for targeting diseased genes. Moreover, the ability toenhance specific characteristics of a protein may also be applicable tochanging the characterized activity of an enzyme to an activitycompletely unrelated to its initially characterized activity. Otherdesirable enhancements of the invention would be specific to eachindividual protein, and would thus be well known in the art andcontemplated by the present invention.

For example, an engineered leucine-rich repeat protein may beconstitutively active upon binding of its cognate ligand. Alternatively,an engineered leucine-rich repeat protein may be constitutively activein the absence of ligand binding. In yet another example, an engineeredleucine-rich repeat protein may be capable of being activated with lessthan all of the regulatory factors and/or conditions typically requiredfor leucine-rich repeat protein activation (e.g., ligand binding,phosphorylation, conformational changes, etc.). Such leucine-rich repeatproteins would be useful in screens to identify leucine-rich repeatprotein modulators, among other uses described herein.

Directed evolution is comprised of several steps. The first step is toestablish a library of variants for the gene or protein of interest. Themost important step is to then select for those variants that entail theactivity you wish to identify. The design of the screen is essentialsince your screen should be selective enough to eliminate non-usefulvariants, but not so stringent as to eliminate all variants. The laststep is then to repeat the above steps using the best variant from theprevious screen. Each successive cycle, can then be tailored asnecessary, such as increasing the stringency of the screen, for example.

Over the years, there have been a number of methods developed tointroduce mutations into macromolecules. Some of these methods include,random mutagenesis, “error-prone” PCR, chemical mutagenesis,site-directed mutagenesis, and other methods well known in the art (fora comprehensive listing of current mutagenesis methods, see Maniatis,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, ColdSpring, N.Y. (1982)). Typically, such methods have been used, forexample, as tools for identifying the core functional region(s) of aprotein or the function of specific domains of a protein (if amulti-domain protein). However, such methods have more recently beenapplied to the identification of macromolecule variants with specific orenhanced characteristics.

Random mutagenesis has been the most widely recognized method to date.Typically, this has been carried out either through the use of“error-prone” PCR (as described in Moore, J., et al, NatureBiotechnology 14:458, (1996), or through the application of randomizedsynthetic oligonucleotides corresponding to specific regions of interest(as described by Derbyshire, K. M. et al, Gene, 46:145-152, (1986), andHill, D E, et al, Methods Enzymol., 55:559-568, (1987). Both approacheshave limits to the level of mutagenesis that can be obtained. However,either approach enables the investigator to effectively control the rateof mutagenesis. This is particularly important considering the fact thatmutations beneficial to the activity of the enzyme are fairly rare. Infact, using too high a level of mutagenesis may counter or inhibit thedesired benefit of a useful mutation.

While both of the aforementioned methods are effective for creatingrandomized pools of macromolecule variants, a third method, termed “DNAShuffling”or “sexual PCR” (WPC, Stemmer, PNAS, 91:10747, (1994)) hasrecently been elucidated. DNA shuffling has also been referred to as“directed molecular evolution”“exon-shuffling”“directed enzymeevolution”“in vitro evolution”and “artificial evolution”. Such referenceterms are known in the art and are encompassed by the invention. Thisnew, preferred, method apparently overcomes the limitations of theprevious methods in that it not only propagates positive traits, butsimultaneously eliminates negative traits in the resulting progeny.

DNA shuffling accomplishes this task by combining the principal of invitro recombination, along with the method of “error-prone” PCR. Ineffect, you begin with a randomly digested pool of small fragments ofyour gene, created by Dnase I digestion, and then introduce said randomfragments into an “error-prone” PCR assembly reaction. During the PCRreaction, the randomly sized DNA fragments not only hybridize to theircognate strand, but also may hybridize to other DNA fragmentscorresponding to different regions of the polynucleotide ofinterest—regions not typically accessible via hybridization of theentire polynucleotide. Moreover, since the PCR assembly reactionutilizes “error-prone” PCR reaction conditions, random mutations areintroduced during the DNA synthesis step of the PCR reaction for all ofthe fragments -further diversifying the potential hybridization sitesduring the annealing step of the reaction.

A variety of reaction conditions could be utilized to carry-out the DNAshuffling reaction. However, specific reaction conditions for DNAshuffling are provided, for example, in PNAS, 91:10747, (1994). Briefly:

Prepare the DNA substrate to be subjected to the DNA shuffling reaction.Preparation may be in the form of simply purifying the DNA fromcontaminating cellular material, chemicals, buffers, oligonucleotideprimers, deoxynucleotides, RNAs, etc., and may entail the use of DNApurification kits as those provided by Qiagen, Inc., or by the Promega,Corp., for example.

Once the DNA substrate has been purified, it would be subjected to DnaseI digestion. About 2-4 ug of the DNA substrate(s) would be digested with0.0015 units of Dnase I (Sigma) per ul in 100 ul of 50 mM Tris-HCL, pH7.4/1 mM MgCl2 for 10-20 min. at room temperature. The resultingfragments of 10-50 bp could then be purified by running them through a2% low-melting point agarose gel by electrophoresis onto DE81ion-exchange paper (Whatmann) or could be purified using Microconconcentrators (Amicon) of the appropriate molecular weight cutoff, orcould use oligonucleotide purification columns (Qiagen), in addition toother methods known in the art. If using DE81 ion-exchange paper, the10-50 bp fragments could be eluted from said paper using 1M NaCl,followed by ethanol precipitation.

The resulting purified fragments would then be subjected to a PCRassembly reaction by re-suspension in a PCR mixture containing: 2 mM ofeach dNTP, 2.2 mM MgCl2, 50 mM KCl, 10 mM Tris●HCL, pH 9.0, and 0.1%Triton X-100, at a final fragment concentration of 10-30 ng/ul. Noprimers are added at this point. Taq DNA polymerase (Promega) would beused at 2.5 units per 100 ul of reaction mixture. A PCR program of 94 Cfor 60 s; 94 C for 30 s, 50-55 C for 30 s, and 72 C for 30 s using 30-45cycles, followed by 72 C for 5 min using an MJ Research (Cambridge,Mass.) PTC-150 thermocycler. After the assembly reaction is completed, a1:40 dilution of the resulting primerless product would then beintroduced into a PCR mixture (using the same buffer mixture used forthe assembly reaction) containing 0.8 um of each primer and subjectingthis mixture to 15 cycles of PCR (using 94 C for 30 s, 50 C for 30 s,and 72 C for 30 s). The referred primers would be primers correspondingto the nucleic acid sequences of the polynucleotide(s) utilized in theshuffling reaction. Said primers could consist of modified nucleic acidbase pairs using methods known in the art and referred to else whereherein, or could contain additional sequences (i.e., for addingrestriction sites, mutating specific base-pairs, etc.).

The resulting shuffled, assembled, and amplified product can be purifiedusing methods well known in the art (e.g., Qiagen PCR purification kits)and then subsequently cloned using appropriate restriction enzymes.

Although a number of variations of DNA shuffling have been published todate, such variations would be obvious to the skilled artisan and areencompassed by the invention. The DNA shuffling method can also betailored to the desired level of mutagenesis using the methods describedby Zhao, et al. (Nucl Acid Res., 25(6):1307-1308, (1997).

As described above, once the randomized pool has been created, it canthen be subjected to a specific screen to identify the variantpossessing the desired characteristic(s). Once the variant has beenidentified, DNA corresponding to the variant could then be used as theDNA substrate for initiating another round of DNA shuffling. This cycleof shuffling, selecting the optimized variant of interest, and thenre-shuffling, can be repeated until the ultimate variant is obtained.Examples of model screens applied to identify variants created using DNAshuffling technology may be found in the following publications: J. C.,Moore, et al., J. Mol. Biol., 272:336-347, (1997), F. R., Cross, et al.,Mol. Cell. Biol., 18:2923-2931, (1998), and A. Crameri., et al., Nat.Biotech., 15:436-438, (1997).

DNA shuffling has several advantages. First, it makes use of beneficialmutations. When combined with screening, DNA shuffling allows thediscovery of the best mutational combinations and does not assume thatthe best combination contains all the mutations in a population.Secondly, recombination occurs simultaneously with point mutagenesis. Aneffect of forcing DNA polymerase to synthesize full-length genes fromthe small fragment DNA pool is a background mutagenesis rate. Incombination with a stringent selection method, enzymatic activity hasbeen evolved up to 16000 fold increase over the wild-type form of theenzyme. In essence, the background mutagenesis yielded the geneticvariability on which recombination acted to enhance the activity.

A third feature of recombination is that it can be used to removedeleterious mutations. As discussed above, during the process of therandomization, for every one beneficial mutation, there may be at leastone or more neutral or inhibitory mutations. Such mutations can beremoved by including in the assembly reaction an excess of the wild-typerandom-size fragments, in addition to the random-size fragments of theselected mutant from the previous selection. During the next selection,some of the most active variants of thepolynucleotide/polypeptide/enzyme, should have lost the inhibitorymutations.

Finally, recombination enables parallel processing. This represents asignificant advantage since there are likely multiple characteristicsthat would make a protein more desirable (e.g. solubility, activity,etc.). Since it is increasingly difficult to screen for more than onedesirable trait at a time, other methods of molecular evolution tend tobe inhibitory. However, using recombination, it would be possible tocombine the randomized fragments of the best representative variants forthe various traits, and then select for multiple properties at once.

DNA shuffling can also be applied to the polynucleotides andpolypeptides of the present invention to decrease their immunogenicityin a specified host. For example, a particular variant of the presentinvention may be created and isolated using DNA shuffling technology.Such a variant may have all of the desired characteristics, though maybe highly immunogenic in a host due to its novel intrinsic structure.Specifically, the desired characteristic may cause the polypeptide tohave a non-native structure which could no longer be recognized as a“self” molecule, but rather as a “foreign”, and thus activate a hostimmune response directed against the novel variant. Such a limitationcan be overcome, for example, by including a copy of the gene sequencefor a xenobiotic ortholog of the native protein in with the genesequence of the novel variant gene in one or more cycles of DNAshuffling. The molar ratio of the ortholog and novel variant DNAs couldbe varied accordingly. Ideally, the resulting hybrid variant identifiedwould contain at least some of the coding sequence which enabled thexenobiotic protein to evade the host immune system, and additionally,the coding sequence of the original novel variant that provided thedesired characteristics.

Likewise, the invention encompasses the application of DNA shufflingtechnology to the evolution of polynucleotides and polypeptides of theinvention, wherein one or more cycles of DNA shuffling include, inaddition to the gene template DNA, oligonucleotides coding for knownallelic sequences, optimized codon sequences, known variant sequences,known polynucleotide polymorphism sequences, known ortholog sequences,known homologue sequences, additional homologous sequences, additionalnon-homologous sequences, sequences from another species, and any numberand combination of the above.

In addition to the described methods above, there are a number ofrelated methods that may also be applicable, or desirable in certaincases. Representative among these are the methods discussed in PCTapplications WO 98/31700, and WO 98/32845, which are hereby incorporatedby reference. Furthermore, related methods can also be applied to thepolynucleotide sequences of the present invention in order to evolveinvention for creating ideal variants for use in gene therapy, proteinengineering, evolution of whole cells containing the variant, or in theevolution of entire enzyme pathways containing polynucleotides of theinvention as described in PCT applications WO 98/13485, WO 98/13487, WO98/27230, WO 98/31837, and Crameri, A., et al., Nat. Biotech.,15:436-438, (1997), respectively.

Additional methods of applying “DNA Shuffling” technology to thepolynucleotides and polypeptides of the present invention, includingtheir proposed applications, may be found in U.S. Pat. No. 5,605,793;PCT Application No. WO 95/22625; PCT Application No. WO 97/20078; PCTApplication No. WO 97/35966; and PCT Application No. WO 98/42832; PCTApplication No. WO 00/09727 specifically provides methods for applyingDNA shuffling to the identification of herbicide selective crops whichcould be applied to the polynucleotides and polypeptides of the presentinvention; additionally, PCT Application No. WO 00/12680 providesmethods and compositions for generating, modifying, adapting, andoptimizing polynucleotide sequences that confer detectable phenotypicproperties on plant species; each of the above are hereby incorporatedin their entirety herein for all purposes.

Example 23 Method of Determining Alterations in a Gene Corresponding toa HLLRCR-1 Polynucleotide

RNA isolated from entire families or individual patients presenting witha phenotype of interest (such as a disease) is be isolated. cDNA is thengenerated from these RNA samples using protocols known in the art. (See,Sambrook.) The cDNA is then used as a template for PCR, employingprimers surrounding regions of interest in SEQ ID NO:1 and/or 3 and/orSEQ ID NO:3. Suggested PCR conditions consist of 35 cycles at 95 degreesC. for 30 seconds; 60-120 seconds at 52-58 degrees C.; and 60-120seconds at 70 degrees C., using buffer solutions described in Sidranskyet al., Science 252:706 (1991).

PCR products are then sequenced using primers labeled at their 5′ endwith T4 polynucleotide kinase, employing SequiTherm Polymerase.(Epicentre Technologies). The intron-exon borders of selected exons isalso determined and genomic PCR products analyzed to confirm theresults. PCR products harboring suspected mutations is then cloned andsequenced to validate the results of the direct sequencing.

PCR products are cloned into T-tailed vectors as described in Holton etal., Nucleic Acids Research, 19:1156 (1991) and sequenced with T7polymerase (United States Biochemical). Affected individuals areidentified by mutations not present in unaffected individuals.

Genomic rearrangements are also observed as a method of determiningalterations in a gene corresponding to a polynucleotide. Genomic clonesisolated according to the methods described herein are nick-translatedwith digoxigenindeoxy-uridine 5′-triphosphate (Boehringer Manheim), andFISH performed as described in Johnson et al., Methods Cell Biol.35:73-99 (1991). Hybridization with the labeled probe is carried outusing a vast excess of human cot-1 DNA for specific hybridization to thecorresponding genomic locus.

Chromosomes are counterstained with 4,6-diamino-2-phenylidole andpropidium iodide, producing a combination of C— and R-bands. Alignedimages for precise mapping are obtained using a triple-band filter set(Chroma Technology, Brattleboro, Vt.) in combination with a cooledcharge-coupled device camera (Photometrics, Tucson, Ariz.) and variableexcitation wavelength filters. (Johnson et al., Genet. Anal. Tech.Appl., 8:75 (1991).) Image collection, analysis and chromosomalfractional length measurements are performed using the ISee GraphicalProgram System. (Inovision Corporation, Durham, N.C.) Chromosomealterations of the genomic region hybridized by the probe are identifiedas insertions, deletions, and translocations. These alterations are usedas a diagnostic marker for an associated disease.

Example 24 Method of Detecting Abnormal Levels of a HLLRCR-1 Polypeptidein a Biological Sample

A polypeptide of the present invention can be detected in a biologicalsample, and if an increased or decreased level of the polypeptide isdetected, this polypeptide is a marker for a particular phenotype.Methods of detection are numerous, and thus, it is understood that oneskilled in the art can modify the following assay to fit theirparticular needs.

For example, antibody-sandwich ELISAs are used to detect polypeptides ina sample, preferably a biological sample. Wells of a microtiter plateare coated with specific antibodies, at a final concentration of 0.2 to10 ug/ml. The antibodies are either monoclonal or polyclonal and areproduced by the method described elsewhere herein. The wells are blockedso that non-specific binding of the polypeptide to the well is reduced.

The coated wells are then incubated for >2 hours at RT with a samplecontaining the polypeptide. Preferably, serial dilutions of the sampleshould be used to validate results. The plates are then washed threetimes with deionized or distilled water to remove unbounded polypeptide.

Next, 50 ul of specific antibody-alkaline phosphatase conjugate, at aconcentration of 25-400 ng, is added and incubated for 2 hours at roomtemperature. The plates are again washed three times with deionized ordistilled water to remove unbounded conjugate.

Add 75 ul of 4-methylumbelliferyl phosphate (MUP) or p-nitrophenylphosphate (NPP) substrate solution to each well and incubate 1 hour atroom temperature. Measure the reaction by a microtiter plate reader.Prepare a standard curve, using serial dilutions of a control sample,and plot polypeptide concentration on the X-axis (log scale) andfluorescence or absorbance of the Y-axis (linear scale). Interpolate theconcentration of the polypeptide in the sample using the standard curve.

Example 25 Formulation of the HLLRCR-1 Polypeptide

The invention also provides methods of treatment and/or preventiondiseases, disorders, and/or conditions (such as, for example, any one ormore of the diseases or disorders disclosed herein) by administration toa subject of an effective amount of a Therapeutic. By therapeutic ismeant a polynucleotides or polypeptides of the invention (includingfragments and variants), agonists or antagonists thereof, and/orantibodies thereto, in combination with a pharmaceutically acceptablecarrier type (e.g., a sterile carrier).

The Therapeutic will be formulated and dosed in a fashion consistentwith good medical practice, taking into account the clinical conditionof the individual patient (especially the side effects of treatment withthe Therapeutic alone), the site of delivery, the method ofadministration, the scheduling of administration, and other factorsknown to practitioners. The “effective amount” for purposes herein isthus determined by such considerations.

As a general proposition, the total pharmaceutically effective amount ofthe Therapeutic administered parenterally per dose will be in the rangeof about 1 ug/kg/day to 10 mg/kg/day of patient body weight, although,as noted above, this will be subject to therapeutic discretion. Morepreferably, this dose is at least 0.01 mg/kg/day, and most preferablyfor humans between about 0.01 and 1 mg/kg/day for the hormone. If givencontinuously, the Therapeutic is typically administered at a dose rateof about 1 ug/kg/hour to about 50 ug/kg/hour, either by 1-4 injectionsper day or by continuous subcutaneous infusions, for example, using amini-pump. An intravenous bag solution may also be employed. The lengthof treatment needed to observe changes and the interval followingtreatment for responses to occur appears to vary depending on thedesired effect.

Therapeutics can be administered orally, rectally, parenterally,intracistemally, intravaginally, intraperitoneally, topically (as bypowders, ointments, gels, drops or transdermal patch), bucally, or as anoral or nasal spray. “Pharmaceutically acceptable carrier” refers to anon-toxic solid, semisolid or liquid filler, diluent, encapsulatingmaterial or formulation auxiliary of any. The term “parenteral” as usedherein refers to modes of administration which include intravenous,intramuscular, intraperitoneal, intrastemal, subcutaneous andintraarticular injection and infusion.

In yet an additional embodiment, the Therapeutics of the invention aredelivered orally using the drug delivery technology described in U.S.Pat. No. 6,258,789, which is hereby incorporated by reference herein.

Therapeutics of the invention are also suitably administered bysustained-release systems. Suitable examples of sustained-releaseTherapeutics are administered orally, rectally, parenterally,intracisternally, intravaginally, intraperitoneally, topically (as bypowders, ointments, gels, drops or transdermal patch), bucally, or as anoral or nasal spray. “Pharmaceutically acceptable carrier” refers to anon-toxic solid, semisolid or liquid filler, diluent, encapsulatingmaterial or formulation auxiliary of any type. The term “parenteral” asused herein refers to modes of administration which include intravenous,intramuscular, intraperitoneal, intrasternal, subcutaneous andintraarticular injection and infusion.

Therapeutics of the invention may also be suitably administered bysustained-release systems. Suitable examples of sustained-releaseTherapeutics include suitable polymeric materials (such as, for example,semi-permeable polymer matrices in the form of shaped articles, e.g.,films, or microcapsules), suitable hydrophobic materials (for example asan emulsion in an acceptable oil) or ion exchange resins, and sparinglysoluble derivatives (such as, for example, a sparingly soluble salt).

Sustained-release matrices include polylactides (U.S. Pat. No.3,773,919, EP 58,481), copolymers of L-glutamic acid andgamma-ethyl-L-glutamate (Sidman et al., Biopolymers 22:547-556 (1983)),poly (2-hydroxyethyl methacrylate) (Langer et al., J. Biomed. Mater.Res. 15:167-277 (1981), and Langer, Chem. Tech. 12:98-105 (1982)),ethylene vinyl acetate (Langer et al., Id.) orpoly-D-(−)-3-hydroxybutyric acid (EP 133,988).

Sustained-release Therapeutics also include liposomally entrappedTherapeutics of the invention (see, generally, Langer, Science249:1527-1533 (1990); Treat et al., in Liposomes in the Therapy ofInfectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss,New York, pp. 317 -327 and 353-365 (1989)). Liposomes containing theTherapeutic are prepared by methods known per se: DE 3,218,121; Epsteinet al., Proc. Natl. Acad. Sci. (USA) 82:3688-3692 (1985); Hwang et al.,Proc. Natl. Acad. Sci.(USA) 77:4030-4034 (1980); EP 52,322; EP 36,676;EP 88,046; EP 143,949; EP 142,641; Japanese Pat. Appl. 83-118008; U.S.Pat. Nos. 4,485,045 and 4,544,545; and EP 102,324. Ordinarily, theliposomes are of the small (about 200-800 Angstroms) unilamellar type inwhich the lipid content is greater than about 30 mol. percentcholesterol, the selected proportion being adjusted for the optimalTherapeutic.

In yet an additional embodiment, the Therapeutics of the invention aredelivered by way of a pump (see Langer, supra; Sefton, CRC Crit. Ref.Biomed. Eng. 14:201 (1987); Buchwald et al., Surgery 88:507 (1980);Saudek et al., N. Engl. J. Med. 321:574 (1989)).

Other controlled release systems are discussed in the review by Langer(Science 249:1527-1533 (1990)).

For parenteral administration, in one embodiment, the Therapeutic isformulated generally by mixing it at the desired degree of purity, in aunit dosage injectable form (solution, suspension, or emulsion), with apharmaceutically acceptable carrier, i.e., one that is non-toxic torecipients at the dosages and concentrations employed and is compatiblewith other ingredients of the formulation. For example, the formulationpreferably does not include oxidizing agents and other compounds thatare known to be deleterious to the Therapeutic.

Generally, the formulations are prepared by contacting the Therapeuticuniformly and intimately with liquid carriers or fmely divided solidcarriers or both. Then, if necessary, the product is shaped into thedesired formulation. Preferably the carrier is a parenteral carrier,more preferably a solution that is isotonic with the blood of therecipient. Examples of such carrier vehicles include water, saline,Ringer's solution, and dextrose solution. Non-aqueous vehicles such asfixed oils and ethyl oleate are also useful herein, as well asliposomes.

The carrier suitably contains minor amounts of additives such assubstances that enhance isotonicity and chemical stability. Suchmaterials are non-toxic to recipients at the dosages and concentrationsemployed, and include buffers such as phosphate, citrate, succinate,acetic acid, and other organic acids or their salts; antioxidants suchas ascorbic acid; low molecular weight (less than about ten residues)polypeptides, e.g., polyarginine or tripeptides; proteins, such as serumalbumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids, such as glycine, glutamic acid,aspartic acid, or arginine; monosaccharides, disaccharides, and othercarbohydrates including cellulose or its derivatives, glucose, mannose,or dextrins; chelating agents such as EDTA; sugar alcohols such asmannitol or sorbitol; counterions such as sodium; and/or nonionicsurfactants such as polysorbates, poloxamers, or PEG.

The Therapeutic will typically be formulated in such vehicles at aconcentration of about 0.1 mg/ml to 100 mg/ml, preferably 1-10 mg/ml, ata pH of about 3 to 8. It will be understood that the use of certain ofthe foregoing excipients, carriers, or stabilizers will result in theformation of polypeptide salts.

Any pharmaceutical used for therapeutic administration can be sterile.Sterility is readily accomplished by filtration through sterilefiltration membranes (e.g., 0.2 micron membranes). Therapeuticsgenerally are placed into a container having a sterile access port, forexample, an intravenous solution bag or vial having a stopper pierceableby a hypodermic injection needle.

Therapeutics ordinarily will be stored in unit or multi-dose containers,for example, sealed ampoules or vials, as an aqueous solution or as alyophilized formulation for reconstitution. As an example of alyophilized formulation, 10-ml vials are filled with 5 ml ofsterile-filtered 1% (w/v) aqueous Therapeutic solution, and theresulting mixture is lyophilized. The infusion solution is prepared byreconstituting the lyophilized Therapeutic using bacteriostaticWater-for-Injection.

The invention also provides a pharmaceutical pack or kit comprising oneor more containers filled with one or more of the ingredients of theTherapeutics of the invention. Associated with such container(s) can bea notice in the form prescribed by a governmental agency regulating themanufacture, use or sale of pharmaceuticals or biological products,which notice reflects approval by the agency of manufacture, use or salefor human administration. In addition, the Therapeutics may be employedin conjunction with other therapeutic compounds.

The Therapeutics of the invention may be administered alone or incombination with adjuvants. Adjuvants that may be administered with theTherapeutics of the invention include, but are not limited to, alum,alum plus deoxycholate (ImmunoAg), MTP-PE (Biocine Corp.), QS21(Genentech, Inc.), BCG, and MPL. In a specific embodiment, Therapeuticsof the invention are administered in combination with alum. In anotherspecific embodiment, Therapeutics of the invention are administered incombination with QS-21. Further adjuvants that may be administered withthe Therapeutics of the invention include, but are not limited to,Monophosphoryl lipid immunomodulator, AdjuVax 100a, QS-21, QS-18,CRL1005, Aluminum salts, MF-59, and Virosomal adjuvant technology.Vaccines that may be administered with the Therapeutics of the inventioninclude, but are not limited to, vaccines directed toward protectionagainst MMR (measles, mumps, rubella), polio, varicella,tetanus/diptheria, hepatitis A, hepatitis B, haemophilus influenzae B,whooping cough, pneumonia, influenza, Lyme's Disease, rotavirus,cholera, yellow fever, Japanese encephalitis, poliomyelitis, rabies,typhoid fever, and pertussis. Combinations may be administered eitherconcomitantly, e.g., as an admixture, separately but simultaneously orconcurrently; or sequentially. This includes presentations in which thecombined agents are administered together as a therapeutic mixture, andalso procedures in which the combined agents are administered separatelybut simultaneously, e.g., as through separate intravenous lines into thesame individual. Administration “in combination” further includes theseparate administration of one of the compounds or agents given first,followed by the second.

The Therapeutics of the invention may be administered alone or incombination with other therapeutic agents. Therapeutic agents that maybe administered in combination with the Therapeutics of the invention,include but not limited to, other members of the TNF family,chemotherapeutic agents, antibiotics, steroidal and non-steroidalanti-inflammatories, conventional immunotherapeutic agents, cytokinesand/or growth factors. Combinations may be administered eitherconcomitantly, e.g., as an admixture, separately but simultaneously orconcurrently; or sequentially. This includes presentations in which thecombined agents are administered together as a therapeutic mixture, andalso procedures in which the combined agents are administered separatelybut simultaneously, e.g., as through separate intravenous lines into thesame individual. Administration “in combination” further includes theseparate administration of one of the compounds or agents given first,followed by the second.

In one embodiment, the Therapeutics of the invention are administered incombination with members of the TNF family. TNF, TNF-related or TNF-likemolecules that may be administered with the Therapeutics of theinvention include, but are not limited to, soluble forms of TNF-alpha,lymphotoxin-alpha (LT-alpha, also known as TNF-beta), LT-beta (found incomplex heterotrimer LT-alpha2-beta), OPGL, FasL, CD27L, CD30L, CD40L,4-1BBL, DcR3, OX40L, TNF-gamma (International Publication No. WO96/14328), AIM-I (International Publication No. WO 97/33899),endokine-alpha (International Publication No. WO 98/07880), TR6(International Publication No. WO 98/30694), OPG, and neutrokine-alpha(International Publication No. WO 98/18921, OX40, and nerve growthfactor (NGF), and soluble forms of Fas, CD30, CD27, CD40 and 4-IBB, TR2(International Publication No. WO 96/34095), DR3 (InternationalPublication No. WO 97/33904), DR4 (International Publication No. WO98/32856), TR5 (International Publication No. WO 98/30693), TR6(International Publication No. WO 98/30694), TR7 (InternationalPublication No. WO 98/41629), TRANK, TR9 (International Publication No.WO 98/56892),TR10 (International Publication No. WO 98/54202), 312C2(International Publication No. WO 98/06842), and TR12, and soluble formsCD154, CD70, and CD153.

In certain embodiments, Therapeutics of the invention are administeredin combination with antiretroviral agents, nucleoside reversetranscriptase inhibitors, non-nucleoside reverse transcriptaseinhibitors, and/or protease inhibitors. Nucleoside reverse transcriptaseinhibitors that may be administered in combination with the Therapeuticsof the invention, include, but are not limited to, RETROVIR(zidovudine/AZT), VIDEX (didanosine/ddI), HIVID (zalcitabine/ddC), ZERIT(stavudine/d4T), EPIVIR (lamivudine/3TC), and COMBIVIR(zidovudine/lamivudine). Non-nucleoside reverse transcriptase inhibitorsthat may be administered in combination with the Therapeutics of theinvention, include, but are not limited to, VIRAMUNE (nevirapine),RESCRIPTOR (delavirdine), and SUSTIVA (efavirenz). Protease inhibitorsthat may be administered in combination with the Therapeutics of theinvention, include, but are not limited to, CRIXIVAN (indinavir), NORVIR(ritonavir), INVIRASE (saquinavir), and VIRACEPT (nelfmavir). In aspecific embodiment, antiretroviral agents, nucleoside reversetranscriptase inhibitors, non-nucleoside reverse transcriptaseinhibitors, and/or protease inhibitors may be used in any combinationwith Therapeutics of the invention to treat AIDS and/or to prevent ortreat HIV infection.

In other embodiments, Therapeutics of the invention may be administeredin combination with anti-opportunistic infection agents.Anti-opportunistic agents that may be administered in combination withthe Therapeutics of the invention, include, but are not limited to,TRIMETHOPRIM-SULFAMETHOXAZOLE, DAPSONE, PENTAMIDINE, ATOVAQUONE,ISONIAZID, RIFAMPIN, PYRAZINAMIDE, ETHAMBUTOL, RIFABUTIN,CLARITHROMYCIN, AZITHROMYCIN, GANCICLOVIR, FOSCARNET, CIDOFOVIR,FLUCONAZOLE, ITRACONAZOLE, KETOCONAZOLE, ACYCLOVIR, FAMCICOLVIR,PYRIMETHAMINE, LEUCOVORIN, NEUPOGEN (filgrastim/G-CSF), and LEUKINE(sargramostim/GM-CSF). In a specific embodiment, Therapeutics of theinvention are used in any combination withTRIMETHOPRIM-SULFAMETHOXAZOLE, DAPSONE, PENTAMIDINE, and/or ATOVAQUONEto prophylactically treat or prevent an opportunistic Pneumocystiscarinii pneumonia infection. In another specific embodiment,Therapeutics of the invention are used in any combination withISONIAZID, RIFAMPIN, PYRAZINAMIDE, and/or ETHAMBUTOL to prophylacticallytreat or prevent an opportunistic Mycobacterium avium complex infection.In another specific embodiment, Therapeutics of the invention are usedin any combination with RIFABUTIN, CLARITHROMYCIN, and/or AZITHROMYCINto prophylactically treat or prevent an opportunistic Mycobacteriumtuberculosis infection. In another specific embodiment, Therapeutics ofthe invention are used in any combination with GANCICLOVIR, FOSCARNET,and/or CIDOFOVIR to prophylactically treat or prevent an opportunisticcytomegalovirus infection. In another specific embodiment, Therapeuticsof the invention are used in any combination with FLUCONAZOLE,ITRACONAZOLE, and/or KETOCONAZOLE to prophylactically treat or preventan opportunistic fungal infection. In another specific embodiment,Therapeutics of the invention are used in any combination with ACYCLOVIRand/or FAMCICOLVIR to prophylactically treat or prevent an opportunisticherpes simplex virus type I and/or type II infection. In anotherspecific embodiment, Therapeutics of the invention are used in anycombination with PYRIMETHAMINE and/or LEUCOVORIN to prophylacticallytreat or prevent an opportunistic Toxoplasma gondii infection. Inanother specific embodiment, Therapeutics of the invention are used inany combination with LEUCOVORIN and/or NEUPOGEN to prophylacticallytreat or prevent an opportunistic bacterial infection.

In a further embodiment, the Therapeutics of the invention areadministered in combination with an antiviral agent. Antiviral agentsthat may be administered with the Therapeutics of the invention include,but are not limited to, acyclovir, ribavirin, amantadine, andremantidine.

In a further embodiment, the Therapeutics of the invention areadministered in combination with an antibiotic agent. Antibiotic agentsthat may be administered with the Therapeutics of the invention include,but are not limited to, amoxicillin, beta-lactamases, aminoglycosides,beta-lactam(glycopeptide), beta-lactamases, Clindamycin,chloramphenicol, cephalosporins, ciprofloxacin, ciprofloxacin,erythromycin, fluoroquinolones, macrolides, metronidazole, penicillins,quinolones, rifampin, streptomycin, sulfonamide, tetracyclines,trimethoprim, trimethoprim-sulfamthoxazole, and vancomycin.

Conventional nonspecific immunosuppressive agents, that may beadministered in combination with the Therapeutics of the inventioninclude, but are not limited to, steroids, cyclosporine, cyclosporineanalogs, cyclophosphamide methylprednisone, prednisone, azathioprine,FK-506, 15-deoxyspergualin, and other immunosuppressive agents that actby suppressing the function of responding T cells.

In specific embodiments, Therapeutics of the invention are administeredin combination with immunosuppressants. Immunosuppressants preparationsthat may be administered with the Therapeutics of the invention include,but are not limited to, ORTHOCLONE (OKT3), SANDIMMUNE/NEORAL/SANGDYA(cyclosporin), PROGRAF (tacrolimus), CELLCEPT (mycophenolate),Azathioprine, glucorticosteroids, and RAPAMUNE (sirolimus). In aspecific embodiment, immunosuppressants may be used to prevent rejectionof organ or bone marrow transplantation.

In an additional embodiment, Therapeutics of the invention areadministered alone or in combination with one or more intravenous immuneglobulin preparations. Intravenous immune globulin preparations that maybe administered with the Therapeutics of the invention include, but notlimited to, GAMMAR, IVEEGAM, SANDOGLOBULIN, GAMMAGARD S/D, and GAMIMUNE.In a specific embodiment, Therapeutics of the invention are administeredin combination with intravenous immune globulin preparations intransplantation therapy (e.g., bone marrow transplant).

In an additional embodiment, the Therapeutics of the invention areadministered alone or in combination with an anti-inflammatory agent.Anti-inflammatory agents that may be administered with the Therapeuticsof the invention include, but are not limited to, glucocorticoids andthe nonsteroidal anti-inflammatories, aminoarylcarboxylic acidderivatives, arylacetic acid derivatives, arylbutyric acid derivatives,arylcarboxylic acids, arylpropionic acid derivatives, pyrazoles,pyrazolones, salicylic acid derivatives, thiazinecarboxamides,e-acetamidocaproic acid, S-adenosylmethionine, 3-amino-4-hydroxybutyricacid, amixetrine, bendazac, benzydamine, bucolome, difenpiramide,ditazol, emorfazone, guaiazulene, nabumetone, nimesulide, orgotein,oxaceprol, paranyline, perisoxal, pifoxime, proquazone, proxazole, andtenidap.

In another embodiment, compositions of the invention are administered incombination with a chemotherapeutic agent. Chemotherapeutic agents thatmay be administered with the Therapeutics of the invention include, butare not limited to, antibiotic derivatives (e.g., doxorubicin,bleomycin, daunorubicin, and dactinomycin); antiestrogens (e.g.,tamoxifen); antimetabolites (e.g., fluorouracil, 5-FU, methotrexate,floxuridine, interferon alpha-2b, glutamic acid, plicamycin,mercaptopurine, and 6-thioguanine); cytotoxic agents (e.g., carmustine,BCNU, lomustine, CCNU, cytosine arabinoside, cyclophosphamide,estramustine, hydroxyurea, procarbazine, mitomycin, busulfan,cis-platin, and vincristine sulfate); hormones (e.g.,medroxyprogesterone, estramustine phosphate sodium, ethinyl estradiol,estradiol, megestrol acetate, methyltestosterone, diethylstilbestroldiphosphate, chlorotrianisene, and testolactone); nitrogen mustardderivatives (e.g., mephalen, chorambucil, mechlorethamine (nitrogenmustard) and thiotepa); steroids and combinations (e.g., bethamethasonesodium phosphate); and others (e.g., dicarbazine, asparaginase,mitotane, vincristine sulfate, vinblastine sulfate, and etoposide).

In a specific embodiment, Therapeutics of the invention are administeredin combination with CHOP (cyclophosphamide, doxorubicin, vincristine,and prednisone) or any combination of the components of CHOP. In anotherembodiment, Therapeutics of the invention are administered incombination with Rituximab. In a further embodiment, Therapeutics of theinvention are administered with Rituxmab and CHOP, or Rituxmab and anycombination of the components of CHOP.

In an additional embodiment, the Therapeutics of the invention areadministered in combination with cytokines. Cytokines that may beadministered with the Therapeutics of the invention include, but are notlimited to, IL2, IL3, IL4, IL5, IL6, IL7, IL10, IL12, IL13, IL15,anti-CD40, CD40L, IFN-gamma and TNF-alpha. In another embodiment,Therapeutics of the invention may be administered with any interleukin,including, but not limited to, IL-1alpha, IL-1beta, IL-2, IL-3, IL-4,IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15,IL-16, IL-17, IL-18, IL-19, IL-20, and IL-21.

In an additional embodiment, the Therapeutics of the invention areadministered in combination with angiogenic proteins. Angiogenicproteins that may be administered with the Therapeutics of the inventioninclude, but are not limited to, Glioma Derived Growth Factor (GDGF), asdisclosed in European Patent Number EP-399816; Platelet Derived GrowthFactor-A (PDGF-A), as disclosed in European Patent Number EP-682110;Platelet Derived Growth Factor-B (PDGF-B), as disclosed in EuropeanPatent Number EP-282317; Placental Growth Factor (P1GF), as disclosed inInternational Publication Number WO 92/06194; Placental Growth Factor-2(P1GF-2), as disclosed in Hauser et al., Gorwth Factors, 4:259-268(1993); Vascular Endothelial Growth Factor (VEGF), as disclosed inInternational Publication Number WO 90/13649; Vascular EndothelialGrowth Factor-A (VEGF-A), as disclosed in European Patent NumberEP-506477; Vascular Endothelial Growth Factor-2 (VEGF-2), as disclosedin International Publication Number WO 96/39515; Vascular EndothelialGrowth Factor B (VEGF-3); Vascular Endothelial Growth Factor B-186(VEGF-B186), as disclosed in International Publication Number WO96/26736; Vascular Endothelial Growth Factor-D (VEGF-D), as disclosed inInternational Publication Number WO 98/02543; Vascular EndothelialGrowth Factor-D (VEGF-D), as disclosed in International PublicationNumber WO 98/07832; and Vascular Endothelial Growth Factor-E (VEGF-E),as disclosed in German Patent Number DE19639601. The above mentionedreferences are incorporated herein by reference herein.

In an additional embodiment, the Therapeutics of the invention areadministered in combination with hematopoietic growth factors.Hematopoietic growth factors that may be administered with theTherapeutics of the invention include, but are not limited to, LEUKINE(SARGRAMOSTIM) and NEUPOGEN (FILGRASTIM).

In an additional embodiment, the Therapeutics of the invention areadministered in combination with Fibroblast Growth Factors. FibroblastGrowth Factors that may be administered with the Therapeutics of theinvention include, but are not limited to, FGF-1, FGF-2, FGF-3, FGF-4,FGF-5, FGF-6, FGF-7, FGF-8, FGF-9, FGF-10, FGF-11, FGF-12, FGF-13,FGF-14, and FGF-15.

In a specific embodiment, formulations of the present invention mayfurther comprise antagonists of P-glycoprotein (also referred to as themultiresistance protein, or PGP), including antagonists of its encodingpolynucleotides (e.g., antisense oligonucleotides, ribozymes, zinc-fmgerproteins, etc.). P-glycoprotein is well known for decreasing theefficacy of various drug administrations due to its ability to exportintracellular levels of absorbed drug to the cell exterior. While thisactivity has been particularly pronounced in cancer cells in response tothe administration of chemotherapy regimens, a variety of other celltypes and the administration of other drug classes have been noted(e.g., T-cells and anti-HIV drugs). In fact, certain mutations in thePGP gene significantly reduces PGP function, making it less able toforce drugs out of cells. People who have two versions of the mutatedgene—one inherited from each paren—have more than four times less PGPthan those with two normal versions of the gene. People may also haveone normal gene and one mutated one. Certain ethnic populations haveincreased incidence of such PGP mutations. Among individuals from Ghana,Kenya, the Sudan, as well as African Americans, frequency of the normalgene ranged from 73% to 84%. In contrast, the frequency was 34% to 59%among British whites, Portuguese, Southwest Asian, Chinese, Filipino andSaudi populations. As a result, certain ethnic populations may requireincreased administration of PGP antagonist in the formulation of thepresent invention to arrive at the an efficacious dose of thetherapeutic (e.g., those from African descent). Conversely, certainethnic populations, particularly those having increased frequency of themutated PGP (e.g., of Caucasian descent, or non-African descent) mayrequire less pharmaceutical compositions in the formulation due to aneffective increase in efficacy of such compositions as a result of theincreased effective absorption (e.g., less PGP activity) of saidcomposition.

Moreover, in another specific embodiment, formulations of the presentinvention may further comprise antagonists of OATP2 (also referred to asthe multiresistance protein, or MRP2), including antagonists of itsencoding polynucleotides (e.g., antisense oligonucleotides, ribozymes,zinc-finger proteins, etc.). The invention also further comprises anyadditional antagonists known to inhibit proteins thought to beattributable to a multidrug resistant phenotype in proliferating cells.

Preferred antagonists that formulations of the present may compriseinclude the potent P-glycoprotein inhibitor elacridar, and/or LY-335979.Other P-glycoprotein inhibitors known in the art are also encompassed bythe present invention.

In additional embodiments, the Therapeutics of the invention areadministered in combination with other therapeutic or prophylacticregimens, such as, for example, radiation therapy.

Example 26 Method of Treating Decreased Levels of the HLLRCR-1Polypeptide

The present invention relates to a method for treating an individual inneed of an increased level of a polypeptide of the invention in the bodycomprising administering to such an individual a composition comprisinga therapeutically effective amount of an agonist of the invention(including polypeptides of the invention). Moreover, it will beappreciated that conditions caused by a decrease in the standard ornormal expression level of a secreted protein in an individual can betreated by administering the polypeptide of the present invention,preferably in the secreted form. Thus, the invention also provides amethod of treatment of an individual in need of an increased level ofthe polypeptide comprising administering to such an individual aTherapeutic comprising an amount of the polypeptide to increase theactivity level of the polypeptide in such an individual.

For example, a patient with decreased levels of a polypeptide receives adaily dose 0.1-100 ug/kg of the polypeptide for six consecutive days.Preferably, the polypeptide is in the secreted form. The exact detailsof the dosing scheme, based on administration and formulation, areprovided herein.

Example 27 Method of Treating Increased Levels of the HLLRCR-1Polypeptide

The present invention also relates to a method of treating an individualin need of a decreased level of a polypeptide of the invention in thebody comprising administering to such an individual a compositioncomprising a therapeutically effective amount of an antagonist of theinvention (including polypeptides and antibodies of the invention).

In one example, antisense technology is used to inhibit production of apolypeptide of the present invention. This technology is one example ofa method of decreasing levels of a polypeptide, preferably a secretedform, due to a variety of etiologies, such as cancer. For example, apatient diagnosed with abnormally increased levels of a polypeptide isadministered intravenously antisense polynucleotides at 0.5, 1.0, 1.5,2.0 and 3.0 mg/kg day for 21 days. This treatment is repeated after a7-day rest period if the treatment was well tolerated. The formulationof the antisense polynucleotide is provided herein.

Example 28 Method of Treatment Using Gene Therapy-Ex Vivo

One method of gene therapy transplants fibroblasts, which are capable ofexpressing a polypeptide, onto a patient. Generally, fibroblasts areobtained from a subject by skin biopsy. The resulting tissue is placedin tissue-culture medium and separated into small pieces. Small chunksof the tissue are placed on a wet surface of a tissue culture flask,approximately ten pieces are placed in each flask. The flask is turnedupside down, closed tight and left at room temperature over night. After24 hours at room temperature, the flask is inverted and the chunks oftissue remain fixed to the bottom of the flask and fresh media (e.g.,Ham's F12 media, with 10% FBS, penicillin and streptomycin) is added.The flasks are then incubated at 37 degree C. for approximately oneweek.

At this time, fresh media is added and subsequently changed everyseveral days. After an additional two weeks in culture, a monolayer offibroblasts emerge. The monolayer is trypsinized and scaled into largerflasks.

pMV-7 (Kirschmeier, P. T. et al., DNA, 7:219-25 (1988)), flanked by thelong terminal repeats of the Moloney murine sarcoma virus, is digestedwith EcoRI and HindIII and subsequently treated with calf intestinalphosphatase. The linear vector is fractionated on agarose gel andpurified, using glass beads.

The cDNA encoding a polypeptide of the present invention can beamplified using PCR primers which correspond to the 5′ and 3′ endsequences respectively as set forth in Example 13 using primers andhaving appropriate restriction sites and initiation/stop codons, ifnecessary. Preferably, the 5′ primer contains an EcoRI site and the 3′primer includes a HindIII site. Equal quantities of the Moloney murinesarcoma virus linear backbone and the amplified EcoRI and HindIIIfragment are added together, in the presence of T4 DNA ligase. Theresulting mixture is maintained under conditions appropriate forligation of the two fragments. The ligation mixture is then used totransform bacteria HB101, which are then plated onto agar containingkanamycin for the purpose of confirming that the vector has the gene ofinterest properly inserted.

The amphotropic pA317 or GP+am12 packaging cells are grown in tissueculture to confluent density in Dulbecco's Modified Eagles Medium (DMEM)with 10% calf serum (CS), penicillin and streptomycin. The MSV vectorcontaining the gene is then added to the media and the packaging cellstransduced with the vector. The packaging cells now produce infectiousviral particles containing the gene (the packaging cells are nowreferred to as producer cells).

Fresh media is added to the transduced producer cells, and subsequently,the media is harvested from a 10 cm plate of confluent producer cells.The spent media, containing the infectious viral particles, is filteredthrough a millipore filter to remove detached producer cells and thismedia is then used to infect fibroblast cells. Media is removed from asub-confluent plate of fibroblasts- and quickly replaced with the mediafrom the producer cells. This media is removed and replaced with freshmedia. If the titer of virus is high, then virtually all fibroblastswill be infected and no selection is required. If the titer is very low,then it is necessary to use a retroviral vector that has a selectablemarker, such as neo or his. Once the fibroblasts have been efficientlyinfected, the fibroblasts are analyzed to determine whether protein isproduced.

The engineered fibroblasts are then transplanted onto the host, eitheralone or after having been grown to confluence on cytodex 3 microcarrierbeads.

Example 29 Gene Therapy Using Endogenous Genes Corresponding to HLLRCR-1Polynucleotides of the Invention

Another method of gene therapy according to the present inventioninvolves operably associating the endogenous polynucleotide sequence ofthe invention with a promoter via homologous recombination as described,for example, in U.S. Pat. No. 5,641,670, issued Jun. 24, 1997;International Publication NO: WO 96/29411, published Sep. 26, 1996;International Publication NO: WO 94/12650, published Aug. 4, 1994;Koller et al., Proc. Natl. Acad. Sci. USA, 86:8932-8935 (1989); andZijlstra et al., Nature, 342:435-438 (1989). This method involves theactivation of a gene which is present in the target cells, but which isnot expressed in the cells, or is expressed at a lower level thandesired.

Polynucleotide constructs are made which contain a promoter andtargeting sequences, which are homologous to the 5′ non-coding sequenceof endogenous polynucleotide sequence, flanking the promoter. Thetargeting sequence will be sufficiently near the 5′ end of thepolynucleotide sequence so the promoter will be operably linked to theendogenous sequence upon homologous recombination. The promoter and thetargeting sequences can be amplified using PCR. Preferably, theamplified promoter contains distinct restriction enzyme sites on the 5′and 3′ ends. Preferably, the 3′ end of the first targeting sequencecontains the same restriction enzyme site as the 5′ end of the amplifiedpromoter and the 5′ end of the second targeting sequence contains thesame restriction site as the 3′ end of the amplified promoter.

The amplified promoter and the amplified targeting sequences aredigested with the appropriate restriction enzymes and subsequentlytreated with calf intestinal phosphatase. The digested promoter anddigested targeting sequences are added together in the presence of T4DNA ligase. The resulting mixture is maintained under conditionsappropriate for ligation of the two fragments. The construct is sizefractionated on an agarose gel then purified by phenol extraction andethanol precipitation.

In this Example, the polynucleotide constructs are administered as nakedpolynucleotides via electroporation. However, the polynucleotideconstructs may also be administered with transfection-facilitatingagents, such as liposomes, viral sequences, viral particles,precipitating agents, etc. Such methods of delivery are known in theart.

Once the cells are transfected, homologous recombination will take placewhich results in the promoter being operably linked to the endogenouspolynucleotide sequence. This results in the expression ofpolynucleotide corresponding to the polynucleotide in the cell.Expression may be detected by immunological staining, or any othermethod known in the art.

Fibroblasts are obtained from a subject by skin biopsy. The resultingtissue is placed in DMEM+10% fetal calf serum. Exponentially growing orearly stationary phase fibroblasts are trypsinized and rinsed from theplastic surface with nutrient medium. An aliquot of the cell suspensionis removed for counting, and the remaining cells are subjected tocentrifugation. The supernatant is aspirated and the pellet isresuspended in 5 ml of electroporation buffer (20 mM HEPES pH 7.3, 137mM NaCl, 5 mM KCl, 0.7 mM Na2 HPO4, 6 mM dextrose). The cells arerecentrifuged, the supernatant aspirated, and the cells resuspended inelectroporation buffer containing 1 mg/ml acetylated bovine serumalbumin. The final cell suspension contains approximately 3×106cells/ml. Electroporation should be performed immediately followingresuspension.

Plasmid DNA is prepared according to standard techniques. For example,to construct a plasmid for targeting to the locus corresponding to thepolynucleotide of the invention, plasmid pUC18 (MBI Fermentas, Amherst,N.Y.) is digested with HindIII. The CMV promoter is amplified by PCRwith an XbaI site on the 5′ end and a BamHI site on the 3′end. Twonon-coding sequences are amplified via PCR: one non-coding sequence(fragment 1) is amplified with a HindlIl site at the 5′ end and an Xbasite at the 3′end; the other non-coding sequence (fragment 2) isamplified with a BamHI site at the 5′end and a HindlIl site at the3′end. The CMV promoter and the fragments (1 and 2) are digested withthe appropriate enzymes (CMV promoter—XbaI and BamHI; fragment 1—XbaI;fragment 2—BamHI) and ligated together. The resulting ligation productis digested with HindIII, and ligated with the HindIII-digested pUC18plasmid.

Plasmid DNA is added to a sterile cuvette with a 0.4 cm electrode gap(Bio-Rad). The final DNA concentration is generally at least 120 μg/ml.0.5 ml of the cell suspension (containing approximately 1.5.×106 cells)is then added to the cuvette, and the cell suspension and DNA solutionsare gently mixed. Electroporation is performed with a Gene-Pulserapparatus (Bio-Rad). Capacitance and voltage are set at 960 ∥F and250-300 V, respectively. As voltage increases, cell survival decreases,but the percentage of surviving cells that stably incorporate theintroduced DNA into their genome increases dramatically. Given theseparameters, a pulse time of approximately 14-20 mSec should be observed.

Electroporated cells are maintained at room temperature forapproximately 5 min, and the contents of the cuvette are then gentlyremoved with a sterile transfer pipette. The cells are added directly to10 ml of prewarmed nutrient media (DMEM with 15% calf serum) in a 10 cmdish and incubated at 37 degree C. The following day, the media isaspirated and replaced with 10 ml of fresh media and incubated for afurther 16-24 hours.

The engineered fibroblasts are then injected into the host, either aloneor after having been grown to confluence on cytodex 3 microcarrierbeads. The fibroblasts now produce the protein product. The fibroblastscan then be introduced into a patient as described above.

Example 30 Method of Treatment Using Gene Therapy—In Vivo

Another aspect of the present invention is using in vivo gene therapymethods to treat disorders, diseases and conditions. The gene therapymethod relates to the introduction of naked nucleic acid (DNA, RNA, andantisense DNA or RNA) sequences into an animal to increase or decreasethe expression of the polypeptide. The polynucleotide of the presentinvention may be operatively linked to a promoter or any other geneticelements necessary for the expression of the polypeptide by the targettissue. Such gene therapy and delivery techniques and methods are knownin the art, see, for example, WO90/11092, WO98/11779; U.S. Pat. Nos.5,693,622, 5,705,151, 5,580,859; Tabata et al., Cardiovasc. Res.35(3):470-479 (1997); Chao et al., Pharmacol. Res. 35(6):517-522 (1997);Wolff, Neuromuscul. Disord. 7(5):314-318 (1997); Schwartz et al., GeneTher. 3(5):405-411 (1996); Tsurumi et al., Circulation 94(12):3281-3290(1996) (incorporated herein by reference).

The polynucleotide constructs may be delivered by any method thatdelivers injectable materials to the cells of an animal, such as,injection into the interstitial space of tissues (heart, muscle, skin,lung, liver, intestine and the like). The polynucleotide constructs canbe delivered in a pharmaceutically acceptable liquid or aqueous carrier.

The term “naked” polynucleotide, DNA or RNA, refers to sequences thatare free from any delivery vehicle that acts to assist, promote, orfacilitate entry into the cell, including viral sequences, viralparticles, liposome formulations, lipofectin or precipitating agents andthe like. However, the polynucleotides of the present invention may alsobe delivered in liposome formulations (such as those taught in FeignerP. L. et al. (1995) Ann. NY Acad. Sci. 772:126-139 and Abdallah B. etal. (1995) Biol. Cell 85(1):1-7) which can be prepared by methods wellknown to those skilled in the art.

The polynucleotide vector constructs used in the gene therapy method arepreferably constructs that will not integrate into the host genome norwill they contain sequences that allow for replication. Any strongpromoter known to those skilled in the art can be used for driving theexpression of DNA. Unlike other gene therapies techniques, one majoradvantage of introducing naked nucleic acid sequences into target cellsis the transitory nature of the polynucleotide synthesis in the cells.Studies have shown that non-replicating DNA sequences can be introducedinto cells to provide production of the desired polypeptide for periodsof up to six months.

The polynucleotide construct can be delivered to the interstitial spaceof tissues within the an animal, including of muscle, skin, brain, lung,liver, spleen, bone marrow, thymus, heart, lymph, blood, bone,cartilage, pancreas, kidney, gall bladder, stomach, intestine, testis,ovary, uterus, rectum, nervous system, eye, gland, and connectivetissue. Interstitial space of the tissues comprises the intercellularfluid, mucopolysaccharide matrix among the reticular fibers of organtissues, elastic fibers in the walls of vessels or chambers, collagenfibers of fibrous tissues, or that same matrix within connective tissueensheathing muscle cells or in the lacunae of bone. It is similarly thespace occupied by the plasma of the circulation and the lymph fluid ofthe lymphatic channels. Delivery to the interstitial space of muscletissue is preferred for the reasons discussed below. They may beconveniently delivered by injection into the tissues comprising thesecells. They are preferably delivered to and expressed in persistent,non-dividing cells which are differentiated, although delivery andexpression may be achieved in non-differentiated or less completelydifferentiated cells, such as, for example, stem cells of blood or skinfibroblasts. In vivo muscle cells are particularly competent in theirability to take up and express polynucleotides.

For the naked polynucleotide injection, an effective dosage amount ofDNA or RNA will be in the range of from about 0.05 g/kg body weight toabout 50 mg/kg body weight. Preferably the dosage will be from about0.005 mg/kg to about 20 mg/kg and more preferably from about 0.05 mg/kgto about 5 mg/kg. Of course, as the artisan of ordinary skill willappreciate, this dosage will vary according to the tissue site ofinjection. The appropriate and effective dosage of nucleic acid sequencecan readily be determined by those of ordinary skill in the art and maydepend on the condition being treated and the route of administration.The preferred route of administration is by the parenteral route ofinjection into the interstitial space of tissues. However, otherparenteral routes may also be used, such as, inhalation of an aerosolformulation particularly for delivery to lungs or bronchial tissues,throat or mucous membranes of the nose. In addition, nakedpolynucleotide constructs can be delivered to arteries duringangioplasty by the catheter used in the procedure.

The dose response effects of injected polynucleotide in muscle in vivois determined as follows. Suitable template DNA for production of mRNAcoding for polypeptide of the present invention is prepared inaccordance with a standard recombinant DNA methodology. The templateDNA, which may be either circular or linear, is either used as naked DNAor complexed with liposomes. The quadriceps muscles of mice are theninjected with various amounts of the template DNA.

Five to six week old female and male Balb/C mice are anesthetized byintraperitoneal injection with 0.3 ml of 2.5% Avertin. A 1.5 cm incisionis made on the anterior thigh, and the quadriceps muscle is directlyvisualized. The template DNA is injected in 0.1 ml of carrier in a 1 ccsyringe through a 27 gauge needle over one minute, approximately 0.5 cmfrom the distal insertion site of the muscle into the knee and about 0.2cm deep. A suture is placed over the injection site for futurelocalization, and the skin is closed with stainless steel clips.

After an appropriate incubation time (e.g., 7 days) muscle extracts areprepared by excising the entire quadriceps. Every fifth 15 urncross-section of the individual quadriceps muscles is histochemicallystained for protein expression. A time course for protein expression maybe done in a similar fashion except that quadriceps from different miceare harvested at different times. Persistence of DNA in muscle followinginjection may be determined by Southern blot analysis after preparingtotal cellular DNA and HIRT supernatants from injected and control mice.The results of the above experimentation in mice can be use toextrapolate proper dosages and other treatment parameters in humans andother animals using naked DNA.

Example 31 Transgenic Animals

The polypeptides of the invention can also be expressed in transgenicanimals. Animals of any species, including, but not limited to, mice,rats, rabbits, hamsters, guinea pigs, pigs, micro-pigs, goats, sheep,cows and non-human primates, e.g., baboons, monkeys, and chimpanzees maybe used to generate transgenic animals. In a specific embodiment,techniques described herein or otherwise known in the art, are used toexpress polypeptides of the invention in humans, as part of a genetherapy protocol.

Any technique known in the art may be used to introduce the transgene(i.e., polynucleotides of the invention) into animals to produce thefounder lines of transgenic animals. Such techniques include, but arenot limited to, pronuclear microinjection (Paterson et al., Appl.Microbiol. Biotechnol. 40:691-698 (1994); Carver et al., Biotechnology(NY) 11:1263-1270 (1993); Wright et al., Biotechnology (NY) 9:830-834(1991); and Hoppe et al., U.S. Pat. No. 4,873,191 (1989)); retrovirusmediated gene transfer into germ lines (Van der Putten et al., Proc.Natl. Acad. Sci., USA 82:6148-6152 (1985)), blastocysts or embryos; genetargeting in embryonic stem cells (Thompson et al., Cell 56:313-321(1989)); electroporation of cells or embryos (Lo, 1983, Mol Cell. Biol.3:1803-1814 (1983)); introduction of the polynucleotides of theinvention using a gene gun (see, e.g., Ulmer et al., Science 259:1745(1993); introducing nucleic acid constructs into embryonic pleuripotentstem cells and transferring the stem cells back into the blastocyst; andsperm-mediated gene transfer (Lavitrano et al., Cell 57:717-723 (1989);etc. For a review of such techniques, see Gordon, “Transgenic Animals,”Intl. Rev. Cytol. 115:171-229 (1989), which is incorporated by referenceherein in its entirety.

Any technique known in the art may be used to produce transgenic clonescontaining polynucleotides of the invention, for example, nucleartransfer into enucleated oocytes of nuclei from cultured embryonic,fetal, or adult cells induced to quiescence (Campell et al., Nature380:64-66 (1996); Wilmut et al., Nature 385:810-813 (1997)).

The present invention provides for transgenic animals that carry thetransgene in all their cells, as well as animals which carry thetransgene in some, but not all their cells, i.e., mosaic animals orchimeric. The transgene may be integrated as a single transgene or asmultiple copies such as in concatamers, e.g., head-to-head tandems orhead-to-tail tandems. The transgene may also be selectively introducedinto and activated in a particular cell type by following, for example,the teaching of Lasko et al. (Lasko et al., Proc. Natl. Acad. Sci. USA89:6232-6236 (1992)). The regulatory sequences required for such acell-type specific activation will depend upon the particular cell typeof interest, and will be apparent to those of skill in the art. When itis desired that the polynucleotide transgene be integrated into thechromosomal site of the endogenous gene, gene targeting is preferred.Briefly, when such a technique is to be utilized, vectors containingsome nucleotide sequences homologous to the endogenous gene are designedfor the purpose of integrating, via homologous recombination withchromosomal sequences, into and disrupting the function of thenucleotide sequence of the endogenous gene. The transgene may also beselectively introduced into a particular cell type, thus inactivatingthe endogenous gene in only that cell type, by following, for example,the teaching of Gu et al. (Gu et al., Science 265:103-106 (1994)). Theregulatory sequences required for such a cell-type specific inactivationwill depend upon the particular cell type of interest, and will beapparent to those of skill in the art.

Once transgenic animals have been generated, the expression of therecombinant gene may be assayed utilizing standard techniques. Initialscreening may be accomplished by Southern blot analysis or PCRtechniques to analyze animal tissues to verify that integration of thetransgene has taken place. The level of mRNA expression of the transgenein the tissues of the transgenic animals may also be assessed usingtechniques which include, but are not limited to, Northern blot analysisof tissue samples obtained from the animal, in situ hybridizationanalysis, and reverse transcriptase-PCR(RT-PCR). Samples of transgenicgene-expressing tissue may also be evaluated immunocytochemically orimmunohistochemically using antibodies specific for the transgeneproduct.

Once the founder animals are produced, they may be bred, inbred,outbred, or crossbred to produce colonies of the particular animal.Examples of such breeding strategies include, but are not limited to:outbreeding of founder animals with more than one integration site inorder to establish separate lines; inbreeding of separate lines in orderto produce compound transgenics that express the transgene at higherlevels because of the effects of additive expression of each transgene;crossing of heterozygous transgenic animals to produce animalshomozygous for a given integration site in order to both augmentexpression and eliminate the need for screening of animals by DNAanalysis; crossing of separate homozygous lines to produce compoundheterozygous or homozygous lines; and breeding to place the transgene ona distinct background that is appropriate for an experimental model ofinterest.

Transgenic animals of the invention have uses which include, but are notlimited to, animal model systems useful in elaborating the biologicalfunction of polypeptides of the present invention, studying diseases,disorders, and/or conditions associated with aberrant expression, and inscreening for compounds effective in ameliorating such diseases,disorders, and/or conditions.

Example 32 Knock-Out Animals

Endogenous gene expression can also be reduced by inactivating or“knocking out” the gene and/or its promoter using targeted homologousrecombination. (E.g., see Smithies et al., Nature 317:230-234 (1985);Thomas & Capecchi, Cell 51:503-512 (1987); Thompson et al., Cell5:313-321 (1989); each of which is incorporated by reference herein inits entirety). For example, a mutant, non-functional polynucleotide ofthe invention (or a completely unrelated DNA sequence) flanked by DNAhomologous to the endogenous polynucleotide sequence (either the codingregions or regulatory regions of the gene) can be used, with or withouta selectable marker and/or a negative selectable marker, to transfectcells that express polypeptides of the invention in vivo. In anotherembodiment, techniques known in the art are used to generate knockoutsin cells that contain, but do not express the gene of interest.Insertion of the DNA construct, via targeted homologous recombination,results in inactivation of the targeted gene. Such approaches areparticularly suited in research and agricultural fields wheremodifications to embryonic stem cells can be used to generate animaloffspring with an inactive targeted gene (e.g., see Thomas & Capecchi1987 and Thompson 1989, supra). However this approach can be routinelyadapted for use in humans provided the recombinant DNA constructs aredirectly administered or targeted to the required site in vivo usingappropriate viral vectors that will be apparent to those of skill in theart.

In further embodiments of the invention, cells that are geneticallyengineered to express the polypeptides of the invention, oralternatively, that are genetically engineered not to express thepolypeptides of the invention (e.g., knockouts) are administered to apatient in vivo. Such cells may be obtained from the patient (i.e.,animal, including human) or an MHC compatible donor and can include, butare not limited to fibroblasts, bone marrow cells, blood cells (e.g.,lymphocytes), adipocytes, muscle cells, endothelial cells etc. The cellsare genetically engineered in vitro using recombinant DNA techniques tointroduce the coding sequence of polypeptides of the invention into thecells, or alternatively, to disrupt the coding sequence and/orendogenous regulatory sequence associated with the polypeptides of theinvention, e.g., by transduction (using viral vectors, and preferablyvectors that integrate the transgene into the cell genome) ortransfection procedures, including, but not limited to, the use ofplasmids, cosmids, YACs, naked DNA, electroporation, liposomes, etc. Thecoding sequence of the polypeptides of the invention can be placed underthe control of a strong constitutive or inducible promoter orpromoter/enhancer to achieve expression, and preferably secretion, ofthe polypeptides of the invention. The engineered cells which expressand preferably secrete the polypeptides of the invention can beintroduced into the patient systemically, e.g., in the circulation, orintraperitoneally.

Alternatively, the cells can be incorporated into a matrix and implantedin the body, e.g., genetically engineered fibroblasts can be implantedas part of a skin graft; genetically engineered endothelial cells can beimplanted as part of a lymphatic or vascular graft. (See, for example,Anderson et al. U.S. Pat. No. 5,399,349; and Mulligan & Wilson, U.S.Pat. No. 5,460,959 each of which is incorporated by reference herein inits entirety).

When the cells to be administered are non-autologous or non-MHCcompatible cells, they can be administered using well known techniqueswhich prevent the development of a host immune response against theintroduced cells. For example, the cells may be introduced in anencapsulated form which, while allowing for an exchange of componentswith the immediate extracellular environment, does not allow theintroduced cells to be recognized by the host immune system.

Transgenic and “knock-out” animals of the invention have uses whichinclude, but are not limited to, animal model systems useful inelaborating the biological function of polypeptides of the presentinvention, studying diseases, disorders, and/or conditions associatedwith aberrant expression, and in screening for compounds effective inameliorating such diseases, disorders, and/or conditions.

Example 33 Method of Isolating Antibody Fragments Directed AgainstHLLRCR-1 From a Library of SCFVS

Naturally occurring V-genes isolated from human PBLs are constructedinto a library of antibody fragments which contain reactivities againstHLLRCR-1 to which the donor may or may not have been exposed (see e.g.,U.S. Pat. No. 5,885,793 incorporated herein by reference in itsentirety).

Rescue of the Library. A library of scFvs is constructed from the RNA ofhuman PBLs as described in PCT publication WO 92/01047. To rescue phagedisplaying antibody fragments, approximately 109 E. coli harboring thephagemid are used to inoculate 50 ml of 2×TY containing 1% glucose and100 μg/ml of ampicillin (2×TY-AMP-GLU) and grown to an O.D. of 0.8 withshaking. Five ml of this culture is used to inoculate 50 ml of2×TY-AMP-GLU, 2×108 TU of delta gene 3 helper (M13 delta gene III, seePCT publication WO 92/01047) are added and the culture incubated at 37°C for 45 minutes without shaking and then at 37° C for 45 minutes withshaking. The culture is centrifuged at 4000 r.p.m. for 10 min. and thepellet resuspended in 2 liters of 2×TY containing 100 μg/ml ampicillinand 50 ug/ml kanamycin and grown overnight. Phage are prepared asdescribed in PCT publication WO 92/01047.

M13 delta gene III is prepared as follows: M13 delta gene III helperphage does not encode gene III protein, hence the phage(mid) displayingantibody fragments have a greater avidity of binding to antigen.Infectious M13 delta gene III particles are made by growing the helperphage in cells harboring a pUC19 derivative supplying the wild type geneIII protein during phage morphogenesis. The culture is incubated for 1hour at 37° C. without shaking and then for a further hour at 37° C.with shaking. Cells are spun down (IEC-Centra 8,400 r.p.m. for 10 min),resuspended in 300 ml 2×TY broth containing 100 μg ampicillin/ml and 25μg kanamycin/ml (2×TY-AMP-KAN) and grown overnight, shaking at 37° C.Phage particles are purified and concentrated from the culture medium bytwo PEG-precipitations (Sambrook et al., 1990), resuspended in 2 ml PBSand passed through a 0.45 μm filter (Minisart NML; Sartorius) to give afinal concentration of approximately 1013 transducing units/ml(ampicillin-resistant clones).

Panning of the Library. Immunotubes (Nunc) are coated overnight in PBSwith 4 ml of either 100 μg/ml or 10 μg/ml of a polypeptide of thepresent invention. Tubes are blocked with 2% Marvel-PBS for 2 hours at37° C. and then washed 3 times in PBS. Approximately 1013 TU of phage isapplied to the tube and incubated for 30 minutes at room temperaturetumbling on an over and under turntable and then left to stand foranother 1.5 hours. Tubes are washed 10 times with PBS 0.1% Tween-20 and10 times with PBS. Phage are eluted by adding 1 ml of 100 mMtriethylamine and rotating 15 minutes on an under and over turntableafter which the solution is immediately neutralized with 0.5 ml of 1.0MTris-HCl, pH 7.4. Phage are then used to infect 10 ml of mid-log E. coliTG1 by incubating eluted phage with bacteria for 30 minutes at 37° C.The E. coli are then plated on TYE plates containing 1% glucose and 100μg/ml ampicillin. The resulting bacterial library is then rescued withdelta gene 3 helper phage as described above to prepare phage for asubsequent round of selection. This process is then repeated for a totalof 4 rounds of affinity purification with tube-washing increased to 20times with PBS, 0.1% Tween-20 and 20 times with PBS for rounds 3 and 4.

Characterization of Binders. Eluted phage from the 3rd and 4th rounds ofselection are used to infect E. coli HB 2151 and soluble scFv isproduced (Marks, et al., 1991) from single colonies for assay. ELISAsare performed with microtitre plates coated with either 10 pg/ml of thepolypeptide of the present invention in 50 mM bicarbonate pH 9.6. Clonespositive in ELISA are further characterized by PCR fingerprinting (see,e.g., PCT publication WO 92/01047) and then by sequencing. These ELISApositive clones may also be further characterized by techniques known inthe art, such as, for example, epitope mapping, binding affinity,receptor signal transduction, ability to block or competitively inhibitantibody/antigen binding, and competitive agonistic. or antagonisticactivity.

Moreover, in another preferred method, the antibodies directed againstthe polypeptides of the present invention may be produced in plants.Specific methods are disclosed in U.S. Pat. Nos. 5,959,177, and6,080,560, which are hereby incorporated in their entirety herein. Themethods not only describe methods of expressing antibodies, but also themeans of assembling foreign multimeric proteins in plants (i.e.,antibodies, etc,), and the subsequent secretion of such antibodies fromthe plant.

Example 34 Identification and Cloning of VH and VL Domains of AntibodiesDirected Against the HLLRCR-1 Polypeptide

VH and VL domains may be identified and cloned from cell linesexpressing an antibody directed against a HLLRCR-1 epitope by performingPCR with VH and VL specific primers on cDNA made from the antibodyexpressing cell lines. Briefly, RNA is isolated from the cell lines andused as a template for RT-PCR designed to amplify the VH and VL domainsof the antibodies expressed by the EBV cell lines. Cells may be lysedusing the TRIzol reagent (Life Technologies, Rockville, Md.) andextracted with one fifth volume of chloroform. After addition ofchloroform, the solution is allowed to incubate at room temperature for10 minutes, and then centrifuged at 14,000 rpm for 15 minutes at 4 C ina tabletop centrifuge. The supernatant is collected and RNA isprecipitated using an equal volume of isopropanol. Precipitated RNA ispelleted by centrifuging at 14,000 rpm for 15 minutes at 4 C in atabletop centrifuge.

Following centrifugation, the supernatant is discarded and washed with75% ethanol. Follwing the wash step, the RNA is centrifuged again at 800rpm for 5 minutes at 4 C. The supernatant is discarded and the pelletallowed to air dry. RNA is the dissolved in DEPC water and heated to 60C for 10 minutes. Quantities of RNA can be determined using opticaldensity measurements. CDNA may be synthesized, according to methodswell-known in the art and/or described herein, from 1.5-2.5 microgramsof RNA using reverse transciptase and random hexamer primers. CDNA isthen used as a template for PCR amplification of VH and VL domains.

Primers used to amplify VH and VL genes are shown below. Typically a PCRreaction makes use of a single 5′primer and a single 3′primer.Sometimes, when the amount of available RNA template is limiting, or forgreater efficiency, groups of 5′ and/or 3′primers may be used. Forexample, sometimes all five VH-5′primers and all JH3′primers are used ina single PCR reaction. The PCR reaction is carried out in a 50microliter volume containing 1× PCR buffer, 2 mM of each dNTP, 0.7 unitsof High Fidelity Taq polymerse, 5′primer mix, 3′primer mix and 7.5microliters of cDNA. The 5′ and 3′primer mix of both VH and VL can bemade by pooling together 22 pmole and 28 pmole, respectively, of each ofthe individual primers. PCR conditions are: 96 C for 5 minutes ;followed by 25 cycles of 94 C for 1 minute, 50 C for 1 minute, and 72 Cfor 1 minute ; followed by an extension cycle of 72 C for 10 minutes.After the reaction has been completed, sample tubes may be stored at 4C. Primer Sequences Used to Amplify VH domains. SEQ ID Primer namePrimer Sequence NO: Hu VH1 - 5′ CAGGTGCAGCTGGTGCAGTCTGG 53 Hu VH2 - 5′CAGGTCAACTTAAGGGAGTCTGG 54 Hu VH3 - 5′ GAGGTGCAGCTGGTGGAGTCTGG 55 HuVH4 - 5′ CAGGTGCAGCTGCAGGAGTCGGG 56 Hu VH5 - 5′ GAGGTGCAGCTGTTGCAGTCTGC57 Hu VH6 - 5′ CAGGTACAGCTGCAGCAGTCAGG 58 Hu JH1 - 5′TGAGGAGACGGTGACCAGGGTGCC 59 Hu JH3 - 5′ TGAAGAGACGGTGACCATTGTCCC 60 HuJH4 - 5′ TGAGGAGACGGTGACCAGGGTTCC 61 Hu JH6 - 5′TGAGGAGACGGTGACCGTGGTCCC 62 Primer Sequences Used to Amplify VL domainsSEQ ID Primer name Primer Sequence NO: Hu Vkappa1 - 5′GACATCCAGATGACCCAGTCTCC 63 Hu Vkappa2a - 5′ GATGTTGTGATGACTCAGTCTCC 64Hu Vkappa2b - 5′ GATATTGTGATGACTCAGTCTCC 65 Hu Vkappa3 - 5′GAAATTGTGTTGACGCAGTCTCC 66 Hu Vkappa4 - 5′ GACATCGTGATGACCCAGTCTCC 67 HuVkappa5 - 5′ GAAACGACACTCACGCAGTCTCC 68 Hu Vkappa6 - 5′GAAATTGTGCTGACTCAGTCTCC 69 Hu Vlambda1 - 5′ CAGTCTGTGTTGACGCAGCCGCC 70Hu Vlambda2 - 5′ CAGTCTGCCCTGACTCAGCCTGC 71 Hu Vlambda3 - 5′TCCTATGTGCTGACTCAGCCACC 72 Hu Vlambda3b - 5′ TCTTCTGAGCTGACTCAGGACCC 73Hu Vlambda4 - 5′ CACGTTATACTGACTCAACCGCC 74 Hu Vlambda5 - 5′CAGGCTGTGCTCACTCAGCCGTC 75 Hu Vlambda6 - 5′ AATTTTATGCTGACTCAGCCCCA 76Hu Jkappa1 - 3′ ACGTTTGATTTCCACCTTGGTCCC 77 Hu Jkappa2 - 3′ACGTTTGATCTCCAGCTTGGTCCC 78 Hu Jkappa3 - 3′ ACGTTTGATATCCACTTTGGTCCC 79Hu Jkappa4 - 3′ ACGTTTGATCTCCACCTTGGTCCC 80 Hu Jkappa5 - 3′ACGTTTAATCTCCAGTCGTGTCCC 81 Hu Vlambda1 - 3′ CAGTCTGTGTTGACGCAGCCGCC 82Hu Vlambda2 - 3′ CAGTCTGCCCTGACTCAGCCTGC 83 Hu Vlambda3 - 3′TCCTATGTGCTGACTCAGCCACC 84 Hu Vlambda3b - 3′ TCTTCTGAGCTGACTCAGGACCC 85Hu Vlambda4 - 3′ CACGTTATACTGACTCAACCGCC 86 Hu Vlambda5 - 3′CAGGCTGTGCTCACTCAGCCGTC 87 Hu Vlambda6 - 3′ AATTTTATGCTGACTCAGCCCCA 88

PCR samples are then electrophoresed on a 1. 3% agarose gel. DNA bandsof the expected sizes (−506 base pairs for VH domains, and 344 basepairs for VL domains) can be cut out of the gel and purified usingmethods well known in the art and/or described herein.

Purified PCR products can be ligated into a PCR cloning vector (TAvector from Invitrogen Inc., Carlsbad, Calfi.A). Individual cloned PCRproducts can be isolated after transfection of E. coli and blue/whitecolor selection. Cloned PCR products may then be sequenced using methodscommonly known in the art and/or described herein.

The PCR bands containing the VH domain and the VL domains can also beused to create full-length Ig expression vectors. VH and VL domains canbe cloned into vectors containing the- nucleotide sequences of a heavy(e.g., human IgGl or human IgG4) or light chain (human kappa or humanambda) constant regions such that a complete heavy or light chainmolecule could be expressed from these vectors when transfected into anappropriate host cell. Further, when cloned heavy and light chains areboth expressed in one cell line (from either one or two vectors), theycan assemble into a complete functional antibody molecule that issecreted into the cell culture medium. Methods using polynucleotidesencoding VH and VL antibody domain to generate expression vectors thatencode complete antibody molecules are well known within the art.

Example 35 Biological Effects of HLLRCR-1 Polypeptides of the InventionAstrocyte and Neuronal Assays

Recombinant polypeptides of the invention, expressed in Escherichia coliand purified as described above, can be tested for activity in promotingthe survival, neurite outgrowth, or phenotypic differentiation ofcortical neuronal cells and for inducing the proliferation of glialfibrillary acidic protein immunopositive cells, astrocytes. Theselection of cortical cells for the bioassay is based on the prevalentexpression of FGF-1 and FGF-2 in cortical structures and on thepreviously reported enhancement of cortical neuronal survival resultingfrom FGF-2 treatment. A thymidine incorporation assay, for example, canbe used to elucidate a polypeptide of the invention's activity on thesecells.

Moreover, previous reports describing the biological effects of FGF-2(basic FGF) on cortical or hippocampal neurons in vitro havedemonstrated increases in both neuron survival and neurite outgrowth(Walicke et al., “Fibroblast growth factor promotes survival ofdissociated hippocampal neurons and enhances neurite extension.” Proc.Natl. Acad. Sci. USA 83:3012-3016. (1986), assay herein incorporated byreference in its entirety). However, reports from experiments done onPC-12 cells suggest that these two responses are not necessarilysynonymous and may depend on not only which FGF is being tested but alsoon which receptor(s) are expressed on the target cells. Using theprimary cortical neuronal culture paradigm, the ability of a polypeptideof the invention to induce neurite outgrowth can be compared to theresponse achieved with FGF-2 using, for example, a thymidineincorporation assay.

Fibroblast and Endothelial Cell Assays

Human lung fibroblasts are obtained from Clonetics (San Diego, Calif.)and maintained in growth media from Clonetics. Dermal microvascularendothelial cells are obtained from Cell Applications (San Diego,Calif.). For proliferation assays, the human lung fibroblasts and dermalmicrovascular endothelial cells can be cultured at 5,000 cells/well in a96-well plate for one day in growth medium. The cells are then incubatedfor one day in 0.1% BSA basal medium. After replacing the medium withfresh 0.1% BSA medium, the cells are incubated with the test proteinsfor 3 days. Alamar Blue (Alamar Biosciences, Sacramento, Calif.) isadded to each well to a final concentration of 10%. The cells areincubated for 4 hr. Cell viability is measured by reading in a CytoFluorfluorescence reader. For the PGE2 assays, the human lung fibroblasts arecultured at 5,000 cells/well in a 96-well plate for one day. After amedium change to 0.1% BSA basal medium, the cells are incubated withFGF-2 or polypeptides of the invention with or without IL-1 ( for 24hours. The supernatants are collected and assayed for PGE2 by EIA kit(Cayman, Ann Arbor, Mich.). For the IL-6 assays, the human lungfibroblasts are cultured at 5,000 cells/well in a 96-well plate for oneday. After a medium change to 0.1% BSA basal medium, the cells areincubated with FGF-2 or with or without polypeptides of the inventionIL-1(for 24 hours. The supernatants are collected and assayed for IL-6by ELISA kit (Endogen, Cambridge, Mass.).

Human lung fibroblasts are cultured with FGF-2 or polypeptides of theinvention for 3 days in basal medium before the addition of Alamar Blueto assess effects on growth of the fibroblasts. FGF-2 should show astimulation at 10-2500 ng/ml which can be used to compare stimulationwith polypeptides of the invention.

Parkinson Models

The loss of motor function in Parkinson's disease is attributed to adeficiency of striatal dopamine resulting from the degeneration of thenigrostriatal dopaminergic projection neurons. An animal model forParkinson's that has been extensively characterized involves thesystemic administration of 1-methyl-4 phenyl 1,2,3,6-tetrahydropyridine(MPTP). In the CNS, MPTP is taken-up by astrocytes and catabolized bymonoamine oxidase B to 1-methyl-4-phenyl pyridine (MPP+) and released.Subsequently, MPP+ is actively accumulated in dopaminergic neurons bythe high-affinity reuptake transporter for dopamine. MPP+ is thenconcentrated in mitochondria by the electrochemical gradient andselectively inhibits nicotidamide adenine disphosphate: ubiquinoneoxidoreductionase (complex I), thereby interfering with electrontransport and eventually generating oxygen radicals.

It has been demonstrated in tissue culture paradigms that FGF-2 (basicFGF) has trophic activity towards nigral dopaminergic neurons (Ferrariet al., Dev. Biol. 1989). Recently, Dr. Unsicker's group hasdemonstrated that administering FGF-2 in gel foam implants in thestriatum results in the near complete protection of nigral dopaminergicneurons from the toxicity associated with MPTP exposure (Otto andUnsicker, J. Neuroscience, 1990).

Based on the data with FGF-2, polypeptides of the invention can beevaluated to determine whether it has an action similar to that of FGF-2in enhancing dopaminergic neuronal survival in vitro and it can also betested in vivo for protection of dopaminergic neurons in the striatumfrom the damage associated with MPTP treatment. The potential effect ofa polypeptide of the invention is first examined in vitro in adopaminergic neuronal cell culture paradigm. The cultures are preparedby dissecting the midbrain floor plate from gestation day 14 Wistar ratembryos. The tissue is dissociated with trypsin and seeded at a densityof 200,000 cells/cm2 on polyorthinine-laminin coated glass coverslips.The cells are maintained in Dulbecco's Modified Eagle's medium and F12medium containing hormonal supplements (N1). The cultures are fixed withparaformaldehyde after 8 days in vitro and are processed for tyrosinehydroxylase, a specific marker for dopaminergic neurons,immunohistochemical staining. Dissociated cell cultures are preparedfrom embryonic rats. The culture medium is changed every third day andthe factors are also added at that time.

Since the dopaminergic neurons are isolated from animals at gestationday 14, a developmental time which is past the stage when thedopaminergic precursor cells are proliferating, an increase in thenumber of tyrosine hydroxylase immunopositive neurons would represent anincrease in the number of dopaminergic neurons surviving in vitro.Therefore, if a polypeptide of the invention acts to prolong thesurvival of dopaminergic neurons, it would suggest that the polypeptidemay be involved in Parkinson's Disease.

One skilled in the art could easily modify the exemplified studies totest the activity of polynucleotides of the invention (e.g., genetherapy), agonists, and/or antagonists of polynucleotides orpolypeptides of the invention.

Example 36 The Effect of the HLLRCR-1 Polypeptides of the Invention onthe Growth of Vascular Endothelial Cells

On day 1, human umbilical vein endothelial cells (HUVEC) are seeded at2-5×104 cells/35 mm dish density in M199 medium containing 4% fetalbovine serum (FBS), 16 units/ml heparin, and 50 units/ml endothelialcell growth supplements (ECGS, Biotechnique, Inc.). On day 2, the mediumis replaced with M199 containing 10% FBS, 8 units/ml heparin. Apolypeptide having the amino acid sequence of SEQ ID NO:2 and/or 4, andpositive controls, such as VEGF and basic FGF (bFGF) are added, atvarying concentrations. On days 4 and 6, the medium is replaced. On day8, cell number is determined with a Coulter Counter.

An increase in the number of HUVEC cells indicates that the polypeptideof the invention may proliferate vascular endothelial cells.

One skilled in the art could easily modify the exemplified studies totest the activity of polynucleotides of the invention (e.g., genetherapy), agonists, and/or antagonists of polynucleotides orpolypeptides of the invention.

Example 37 Stimulatory Effect of HLLRCR-1 Polypeptides of the Inventionon the Proliferation of Vascular Endothelial Cells

For evaluation of mitogenic activity of growth factors, the colorimetricMTS(3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)2H-tetrazolium)assay with the electron coupling reagent PMS (phenazine methosulfate)was performed (CellTiter 96 AQ, Promega). Cells are seeded in a 96-wellplate (5,000 cells/well) in 0.1 mL serum-supplemented medium and areallowed to attach overnight. After serum-starvation for 12 hours in 0.5%FBS, conditions (bFGF, VEGF165 or a polypeptide of the invention in 0.5%FBS) with or without Heparin (8 U/ml) are added to wells for 48 hours.20 mg of MTS/PMS mixture (1:0.05) are added per well and allowed toincubate for 1 hour at 37° C. before measuring the absorbance at 490 nmin an ELISA plate reader. Background absorbance from control wells (somemedia, no cells) is subtracted, and seven wells are performed inparallel for each condition. See, Leak et al. In Vitro Cell. Dev. Biol.30A:512-518 (1994).

One skilled in the art could easily modify the exemplified studies totest the activity of polynucleotides of the invention (e.g., genetherapy), agonists, and/or antagonists of polynucleotides orpolypeptides of the invention.

Example 38 Inhibition of PDGF-Induced Vascular Smooth Muscle CellProliferation Stimulatory Effect

HAoSMC proliferation can be measured, for example, by BrdUrdincorporation. Briefly, subconfluent, quiescent cells grown on the4-chamber slides are transfected with CRP or FITC-labeled AT2-3LP. Then,the cells are pulsed with 10% calf serum and 6 mg/ml BrdUrd. After 24 h,immunocytochemistry is performed by using BrdUrd Staining Kit (ZymedLaboratories). In brief, the cells are incubated with the biotinylatedmouse anti-BrdUrd antibody at 4 degrees C for 2 h after being exposed todenaturing solution and then incubated with the streptavidin-peroxidaseand diaminobenzidine. After counterstaining with hematoxylin, the cellsare mounted for microscopic examination, and the BrdUrd-positive cellsare counted. The BrdUrd index is calculated as a percent of theBrdUrd-positive cells to the total cell number. In addition, thesimultaneous detection of the BrdUrd staining (nucleus) and the FITCuptake (cytoplasm) is performed for individual cells by the concomitantuse of bright field illumination and dark field-UV fluorescentillumination. See, Hayashida et al., J. Biol. Chem.6:271(36):21985-21992 (1996).

One skilled in the art could easily modify the exemplified studies totest the activity of polynucleotides of the invention (e.g., genetherapy), agonists, and/or antagonists of polynucleotides orpolypeptides of the invention.

Example 39 Stimulation of Endothelial Migration

This example will be used to explore the possibility that a polypeptideof the invention may stimulate lymphatic endothelial cell migration.

Endothelial cell migration assays are performed using a 48 wellmicrochemotaxis chamber (Neuroprobe Inc., Cabin John, M D; Falk, W., etal., J. Immunological Methods 1980;33:239-247).Polyvinylpyrrolidone-free polycarbonate filters with a pore size of 8 um(Nucleopore Corp. Cambridge, Mass.) are coated with 0.1% gelatin for atleast 6 hours at room temperature and dried under sterile air. Testsubstances are diluted to appropriate concentrations in M199supplemented with 0.25% bovine serum albumin (BSA), and 25 ul of thefinal dilution is placed in the lower chamber of the modified Boydenapparatus. Subconfluent, early passage (2-6) HUVEC or BMEC cultures arewashed and trypsinized for the minimum time required to achieve celldetachment. After placing the filter between lower and upper chamber,2.5×105 cells suspended in 50 ul M199 containing 1% FBS are seeded inthe upper compartment. The apparatus is then incubated for 5 hours at37° C. in a humidified chamber with 5% CO2 to allow cell migration.After the incubation period, the filter is removed and the upper side ofthe filter with the non-migrated cells is scraped with a rubberpoliceman. The filters are fixed with methanol and stained with a Giemsasolution (Diff-Quick, Baxter, McGraw Park, Ill.). Migration isquantified by counting cells of three random high-power fields (40×) ineach well, and all groups are performed in quadruplicate.

One skilled in the art could easily modify the exemplified studies totest the activity of polynucleotides of the invention (e.g., genetherapy), agonists, and/or antagonists of polynucleotides orpolypeptides of the invention.

Example 40 Stimulation of Nitric Oxide Productoin by Endothelial Cells

Nitric oxide released by the vascular endothelium is believed to be amediator of vascular endothelium relaxation. Thus, activity of apolypeptide of the invention can be assayed by determining nitric oxideproduction by endothelial cells in response to the polypeptide.

Nitric oxide is measured in 96-well plates of confluent microvascularendothelial cells after 24 hours starvation and a subsequent 4 hrexposure to various levels of a positive control (such as VEGF-1) andthe polypeptide of the invention. Nitric oxide in the medium isdetermined by use of the Griess reagent to measure total nitrite afterreduction of nitric oxide-derived nitrate by nitrate reductase. Theeffect of the polypeptide of the invention on nitric oxide release isexamined on HUVEC.

Briefly, NO release from cultured HUVEC monolayer is measured with aNO-specific polarographic electrode connected to a NO meter (Iso-NO,World Precision Instruments Inc.) (1049). Calibration of the NO elementsis performed according to the following equation:2 KNO2+2 KI+2 H2SO4 6 2 NO+I2+2 H2O+2 K2SO4

The standard calibration curve is obtained by adding gradedconcentrations of KNO2 (0, 5, 10, 25, 50, 100, 250, and 500 nmol/L) intothe calibration solution containing KI and H2SO4. The specificity of theIso-NO electrode to NO is previously determined by measurement of NOfrom authentic NO gas (1050). The culture medium is removed and HUVECsare washed twice with Dulbecco's phosphate buffered saline. The cellsare then bathed in 5 ml of filtered Krebs-Henseleit solution in 6-wellplates, and the cell plates are kept on a slide warmer (Lab LineInstruments Inc.) To maintain the temperature at 37° C. The NO sensorprobe is inserted vertically into the wells, keeping the tip of theelectrode 2 mm under the surface of the solution, before addition of thedifferent conditions. S-nitroso acetyl penicillamin (SNAP) is used as apositive control. The amount of released NO is expressed as picomolesper 1×106 endothelial cells. All values reported are means of four tosix measurements in each group (number of cell culture wells). See, Leaket al. Biochem. and Biophys. Res. Comm. 217:96-105 (1995).

One skilled in the art could easily modify the exemplified studies totest the activity of polynucleotides of the invention (e.g., genetherapy), agonists, and/or antagonists of polynucleotides orpolypeptides of the invention.

Example 41 Effect of HLLRCR-1 Polypeptides of the Invention onVasodilation

Since dilation of vascular endothelium is important in reducing bloodpressure, the ability of polypeptides of the invention to affect theblood pressure in spontaneously hypertensive rats (SHR) is examined.Increasing doses (0, 10, 30, 100, 300, and 900 mg/kg) of thepolypeptides of the invention are administered to 13-14 week oldspontaneously hypertensive rats (SHR). Data are expressed as the mean±SEM. Statistical analysis are performed with a paired t-test andstatistical significance is defined as p<0.05 vs. the response to bufferalone.

One skilled in the art could easily modify the exemplified studies totest the activity of polynucleotides of the invention (e.g., genetherapy), agonists, and/or antagonists of polynucleotides orpolypeptides of the invention.

Example 42 Peripheral Arterial Disease Model

Angiogenic therapy using a polypeptide of the invention is a noveltherapeutic strategy to obtain restoration of blood flow around theischemia in case of peripheral arterial diseases. The experimentalprotocol includes:

(a) One side of the femoral artery is ligated to create ischemic muscleof the hindlimb, the other side of hindlimb serves as a control.

(b) a polypeptide of the invention, in a dosage range of 20 mg-500 mg,is delivered intravenously and/or intramuscularly 3 times (perhaps more)per week for 2-3 weeks.

(c) The ischemic muscle tissue is collected after ligation of thefemoral artery at 1, 2, and 3 weeks for the analysis of expression of apolypeptide of the invention and histology. Biopsy is also performed onthe other side of normal muscle of the contralateral hindlimb.

One skilled in the art could easily modify the exemplified studies totest the activity of polynucleotides of the invention (e.g., genetherapy), agonists, and/or antagonists of polynucleotides orpolypeptides of the invention.

Example 43 Ischemic Myocardial Disease Model

A polypeptide of the invention is evaluated as a potent mitogen capableof stimulating the development of collateral vessels, and restructuringnew vessels after coronary artery occlusion. Alteration of expression ofthe polypeptide is investigated in situ. The experimental protocolincludes:

(a) The heart is exposed through a left-side thoracotomy in the rat.Immediately, the left coronary artery is occluded with a thin suture(6-0) and the thorax is closed.

(b) a polypeptide of the invention, in a dosage range of 20 mg-500 mg,is delivered intravenously and/or intramuscularly 3 times (perhaps more)per week for 2-4 weeks.

(c) Thirty days after the surgery, the heart is removed andcross-sectioned for morphometric and in situ analyzes.

One skilled in the art could easily modify the exemplified studies totest the activity of polynucleotides of the invention (e.g., genetherapy), agonists, and/or antagonists of polynucleotides orpolypeptides of the invention.

Example 44 Suppression of the Alpha-Induced Adhesion Molecule Expressionby a HLLRCR-1 Polypeptide of the Invention

The recruitment of lymphocytes to areas of inflammation and angiogenesisinvolves specific receptor-ligand interactions between cell surfaceadhesion molecules (CAMs) on lymphocytes and the vascular endothelium.The adhesion process, in both normal and pathological settings, followsa multi-step cascade that involves intercellular adhesion molecule-1(ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and endothelialleukocyte adhesion molecule-1 (E-selectin) expression on endothelialcells (EC). The expression of these molecules and others on the vascularendothelium determines the efficiency with which leukocytes may adhereto the local vasculature and extravasate into the local tissue duringthe development of an inflammatory response. The local concentration ofcytokines and growth factor participate in the modulation of theexpression of these CAMs.

Tumor necrosis factor alpha (TNF-a), a potent proinflammatory cytokine,is a stimulator of all three CAMs on endothelial cells and may beinvolved in a wide variety of inflammatory responses, often resulting ina pathological outcome.

The potential of a polypeptide of the invention to mediate a suppressionof TNF-a induced CAM expression can be examined. A modified ELISA assaywhich uses ECs as a solid phase absorbent is employed to measure theamount of CAM expression on TNF-a treated ECs when co-stimulated with amember of the FGF family of proteins.

To perform the experiment, human umbilical vein endothelial cell (HUVEC)cultures are obtained from pooled cord harvests and maintained in growthmedium (EGM-2; Clonetics, San Diego, Calif.) supplemented with 10% FCSand 1% penicillin/streptomycin in a 37 degree C. humidified incubatorcontaining 5% CO2. HUVECs are seeded in 96-well plates at concentrationsof 1×104 cells/well in EGM medium at 37 degree C. for 18-24 hrs or untilconfluent. The monolayers are subsequently washed 3 times with aserum-free solution of RPMI-1640 supplemented with 100 U/ml penicillinand 100 mg/ml streptomycin, and treated with a given cytokine and/orgrowth factor(s) for 24 h at 37 degree C. Following incubation, thecells are then evaluated for CAM expression.

Human Umbilical Vein Endothelial cells (HUVECs) are grown in a standard96 well plate to confluence. Growth medium is removed from the cells andreplaced with 90 ul of 199 Medium (10% FBS). Samples for testing andpositive or negative controls are added to the plate in triplicate (in10 ul volumes). Plates are incubated at 37 degree C. for either 5 h(selectin and integrin expression) or 24 h (integrin expression only).Plates are aspirated to remove medium and 100 μl of 0.1%paraformaldehyde-PBS(with Ca++ and Mg++) is added to each well. Platesare held at 4° C. for 30 min.

Fixative is then removed from the wells and wells are washed 1× withPBS(+Ca,Mg)+0.5% BSA and drained. Do not allow the wells to dry. Add 10μl of diluted primary antibody to the test and control wells.Anti-ICAM-1-Biotin, Anti-VCAM-1-Biotin and Anti-E-selectin-Biotin areused at a concentration of 10 μg/ml (1:10 dilution of 0.1 mg/ml stockantibody). Cells are incubated at 37° C. for 30 min. in a humidifiedenvironment. Wells are washed X3 with PBS(+Ca,Mg)+0.5% BSA.

Then add 20 μl of diluted ExtrAvidin-Alkaline Phosphatase (1:5,000dilution) to each well and incubated at 37° C. for 30 min. Wells arewashed X3 with PBS(+Ca,Mg)+0.5% BSA. 1 tablet of p-Nitrophenol PhosphatepNPP is dissolved in 5 ml of glycine buffer (pH 10.4). 100 μl of pNPPsubstrate in glycine buffer is added to each test well. Standard wellsin triplicate are prepared from the working dilution of theExtrAvidin-Alkaline Phosphatase in glycine buffer: 1:5,000(100) >10-0.5>10-1>10-1.5. 5 μl of each dilution is added to triplicatewells and the resulting AP content in each well is 5.50 ng, 1.74 ng,0.55 ng, 0.18 ng. 100 pl of pNNP reagent must then be added to each ofthe standard wells. The plate must be incubated at 37° C. for 4 h. Avolume of 50 μl of 3M NaOH is added to all wells. The results arequantified on a plate reader at 405 nm. The background subtractionoption is used on blank wells filled with glycine buffer only. Thetemplate is set up to indicate the concentration of AP-conjugate in eachstandard well [5.50 ng; 1.74 ng; 0.55 ng; 0.18 ng]. Results areindicated as amount of bound AP-conjugate in each sample.

One skilled in the art could easily modify the exemplified studies totest the activity of polynucleotides of the invention (e.g., genetherapy), agonists, and/or antagonists of polynucleotides orpolypeptides of the invention.

Example 56 Method of Creating N— and C-Terminal Deletion MutantsCorresponding to the HLRRCR-1 Polypeptide of the Present Invention

As described elsewhere herein, the present invention encompasses thecreation of N— and C-terminal deletion mutants, in addition to anycombination of N— and C-terminal deletions thereof, corresponding to theHLRRCR-1 polypeptide of the present invention. A number of methods areavailable to one skilled in the art for creating such mutants. Suchmethods may include a combination of PCR amplification and gene cloningmethodology. Although one of skill in the art of molecular biology,through the use of the teachings provided or referenced herein, and/orotherwise known in the art as standard methods, could readily createeach deletion mutant of the present invention, exemplary methods aredescribed below.

Briefly, using the isolated cDNA clone encoding the full-length HLRRCR-1polypeptide sequence (as described in Example 14, for example),appropriate primers of about 15-25 nucleotides derived from the desired5′ and 3′ positions of SEQ ID NO:1 and/or 3 may be designed to PCRamplify, and subsequently clone, the intended N— and/or C-terminaldeletion mutant. Such primers could comprise, for example, aninititation and stop codon for the 5′ and 3′ primer, respectively. Suchprimers may also comprise restriction sites to facilitate cloning of thedeletion mutant post amplification. Moreover, the primers may compriseadditional sequences, such as, for example, flag-tag sequences, kozacsequences, or other sequences discussed and/or referenced herein.

For example, in the case of the D290 to H517 N-terminal deletion mutant,the following primers could be used to amplify a cDNA fragmentcorresponding to this deletion mutant: 5′ 5′-GCAGCAGCGGCCGC CCAGATGTGGCCCTGCTGACTGTCC-3′ Primer             NotI (SEQ IDNO:47) 3′ 5′-GCAGCA GTCGAC GTGTAAGCCATTGTCTTCTTTTTC-3′ Primer           SalI (SEQ ID NO:48)

For example, in the case of the M1 to F313 C-terminal deletion mutant,the following primers could be used to amplify a cDNA fragmentcorresponding to this deletion mutant: 5′ 5′-GCAGCAGCGGCCGC ATGTTGCGGTTGGTGGCAGCTTGCC-3′ Primer             NotI (SEQ IDNO:49) 3′ 5′-GCAGCA GTCGAC TACAACTAAACCTAGGCAAGTGAG-3′ Primer            SalI (SEQ ID NO:50)

Representative PCR amplification conditions are provided below, althoughthe skilled artisan would appreciate that other conditions may berequired for efficient amplification. A 100 ul PCR reaction mixture maybe prepared using 10 ng of the template DNA (cDNA clone of HLRRCR-1),200 uM 4dNTPs, 1 uM primers, 0.25U Taq DNA polymerase (PE), and standardTaq DNA polymerase buffer. Typical PCR cycling condition are as follows:

20-25 cycles: 45 sec, 93 degrees

-   -   2 min, 50 degrees    -   2 min, 72 degrees

1 cycle: 10 min, 72 degrees

After the final extension step of PCR, 5U Klenow Fragment may be addedand incubated for 15 min at 30 degrees.

Upon digestion of the fragment with the NotI and SalI restrictionenzymes, the fragment could be cloned into an appropriate expressionand/or cloning vector which has been similarly digested (e.g., pSport1,among others). . The skilled artisan would appreciate that otherplasmids could be equally substituted, and may be desirable in certaincircumstances. The digested fragment and vector are then ligated using aDNA ligase, and then used to transform competent E. coli cells usingmethods provided herein and/or otherwise known in the art.

The 5′ primer sequence for amplifying any additional N-terminal deletionmutants may be determined by reference to the following formula:

(S+(X*3)) to ((S+(X*3))+25), wherein ‘S’ is equal to the nucleotideposition of the initiating start codon of the HLRRCR-1 gene (SEQ ID NO:1and/or 3), and ‘X’ is equal to the most N-terminal amino acid of theintended N-terminal deletion mutant. The first term will provide thestart 5′ nucleotide position of the 5′ primer, while the second termwill provide the end 3′ nucleotide position of the 5′ primercorresponding to sense strand of SEQ ID NO:1 and/or 3. Once thecorresponding nucleotide positions of the primer are determined, thefinal nucleotide sequence may be created by the addition of applicablerestriction site sequences to the 5′ end of the sequence, for example.As referenced herein, the addition of other sequences to the 5′ primermay be desired in certain circumstances (e.g., kozac sequences, etc.).

The 3′ primer sequence for amplifying any additional N-terminal deletionmutants may be determined by reference to the following formula:

(S+(X*3)) to ((S+(X*3))−25), wherein ‘S’ is equal to the nucleotideposition of the initiating start codon of the HLRRCR-1 gene (SEQ ID NO:1and/or 3), and ‘X’ is equal to the most C-terminal amino acid of theintended N-terminal deletion mutant. The first term will provide thestart 5′ nucleotide position of the 3′ primer, while the second termwill provide the end 3′ nucleotide position of the 3′ primercorresponding to the anti-sense strand of SEQ ID NO:1 and/or 3. Once thecorresponding nucleotide positions of the primer are determined, thefinal nucleotide sequence may be created by the addition of applicablerestriction site sequences to the 5′ end of the sequence, for example.As referenced herein, the addition of other sequences to the 3′ primermay be desired in certain circumstances (e.g., stop codon sequences,etc.). The skilled artisan would appreciate that modifications of theabove nucleotide positions may be necessary for optimizing PCRamplification.

The same general formulas provided above may be used in identifying the5′ and 3′ primer sequences for amplifying any C-terminal deletion mutantof the present invention. Moreover, the same general formulas providedabove may be used in identifying the 5′ and 3′ primer sequences foramplifying any combination of N— terminal and C-terminal deletion mutantof the present invention. The skilled artisan would appreciate thatmodifications of the above nucleotide positions may be necessary foroptimizing PCR amplification.

Example 57 Method of Enhancing the Biological Activity/FunctionalCharacteristics of Invention Through Molecular Evolution

Although many of the most biologically active proteins known are highlyeffective for their specified function in an organism, they oftenpossess characteristics that make them undesirable for transgenic,therapeutic, pharmaceutical, and/or industrial applications. Among thesetraits, a short physiological half-life is the most prominent problem,and is present either at the level of the protein, or the level of theproteins mRNA. The ability to extend the half-life, for example, wouldbe particularly important for a proteins use in gene therapy, transgenicanimal production, the bioprocess production and purification of theprotein, and use of the protein as a chemical modulator among others.Therefore, there is a need to identify novel variants of isolatedproteins possessing characteristics which enhance their application as atherapeutic for treating diseases of animal origin, in addition to theproteins applicability to common industrial and pharmaceuticalapplications.

Thus, one aspect of the present invention relates to the ability toenhance specific characteristics of invention through directed molecularevolution. Such an enhancement may, in a non-limiting example, benefitthe inventions utility as an essential component in a kit, theinventions physical attributes such as its solubility, structure, orcodon optimization, the inventions specific biological activity,including any associated enzymatic activity, the proteins enzymekinetics, the proteins Ki, Kcat, Km, Vmax, Kd, protein-protein activity,protein-DNA binding activity, antagonist/inhibitory activity (includingdirect or indirect interaction), agonist activity (including direct orindirect interaction), the proteins antigenicity (e.g., where it wouldbe desirable to either increase or decrease the antigenic potential ofthe protein), the immunogenicity of the protein, the ability of theprotein to form dimers, trimers, or multimers with either itself orother proteins, the antigenic efficacy of the invention, including itssubsequent use a preventative treatment for disease or disease states,or as an effector for targeting diseased genes. Moreover, the ability toenhance specific characteristics of a protein may also be applicable tochanging the characterized activity of an enzyme to an activitycompletely unrelated to its initially characterized activity. Otherdesirable enhancements of the invention would be specific to eachindividual protein, and would thus be well known in the art andcontemplated by the present invention.

For example, an engineered leucine-rich repeat protein may beconstitutively active upon binding of its cognate ligand. Alternatively,an engineered leucine-rich repeat protein may be constitutively activein the absence of ligand binding. In yet another example, an engineeredleucine-rich repeat protein may be capable of being activated with lessthan all of the regulatory factors and/or conditions typically requiredfor leucine-rich repeat protein activation (e.g., ligand binding,phosphorylation, conformational changes, etc.). Such leucine-rich repeatprotein would be useful in screens to identify leucine-rich repeatprotein modulators, among other uses described herein.

Directed evolution is comprised of several steps. The first step is toestablish a library of variants for the gene or protein of interest. Themost important step is to then select for those variants that entail theactivity you wish to identify. The design of the screen is essentialsince your screen should be selective enough to eliminate non-usefulvariants, but not so stringent as to eliminate all variants. The laststep is then to repeat the above steps using the best variant from theprevious screen. Each successive cycle, can then be tailored asnecessary, such as increasing the stringency of the screen, for example.

Over the years, there have been a number of methods developed tointroduce mutations into macromolecules. Some of these methods include,random mutagenesis, “error-prone” PCR, chemical mutagenesis,site-directed mutagenesis, and other methods well known in the art (fora comprehensive listing of current mutagenesis methods, see Maniatis,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, ColdSpring, N.Y. (1982)). Typically, such methods have been used, forexample, as tools for identifying the core functional region(s) of aprotein or the function of specific domains of a protein (if amulti-domain protein). However, such methods have more recently beenapplied to the identification of macromolecule variants with specific orenhanced characteristics.

Random mutagenesis has been the most widely recognized method to date.Typically, this has been carried out either through the use of“error-prone” PCR (as described in Moore, J., et al, NatureBiotechnology 14:458, (1996), or through the application of randomizedsynthetic oligonucleotides corresponding to specific regions of interest(as described by Derbyshire, K. M. et al, Gene, 46:145-152, (1986), andHill, D E, et al, Methods Enzymol., 55:559-568, (1987). Both approacheshave limits to the level of mutagenesis that can be obtained. However,either approach enables the investigator to effectively control the rateof mutagenesis. This is particularly important considering the fact thatmutations beneficial to the activity of the enzyme are fairly rare. Infact, using too high a level of mutagenesis may counter or inhibit thedesired benefit of a useful mutation.

While both of the aforementioned methods are effective for creatingrandomized pools of macromolecule variants, a third method, termed “DNAShuffling”or “sexual PCR” (WPC, Stemmer, PNAS, 91:10747, (1994)) hasrecently been elucidated. DNA shuffling has also been referred to as“directed molecular evolution”“exon-shuffling”“directed enzymeevolution”“in vitro evolution”and “artificial evolution”. Such referenceterms are known in the art and are encompassed by the invention. Thisnew, preferred, method apparently overcomes the limitations of theprevious methods in that it not only propagates positive traits, butsimultaneously eliminates negative traits in the resulting progeny.

DNA shuffling accomplishes this task by combining the principal of invitro recombination, along with the method of “error-prone” PCR. Ineffect, you begin with a randomly digested pool of small fragments ofyour gene, created by Dnase I digestion, and then introduce said randomfragments into an “error-prone” PCR assembly reaction. During the PCRreaction, the randomly sized DNA fragments not only hybridize to theircognate strand, but also may hybridize to other DNA fragmentscorresponding to different regions of the polynucleotide ofinterest—regions not typically accessible via hybridization of theentire polynucleotide. Moreover, since the PCR assembly reactionutilizes “error-prone” PCR reaction conditions, random mutations areintroduced during the DNA synthesis step of the PCR reaction for all ofthe fragments -further diversifying the potential hybridization sitesduring the annealing step of the reaction.

A variety of reaction conditions could be utilized to carry-out the DNAshuffling reaction. However, specific reaction conditions for DNAshuffling are provided, for example, in PNAS, 91:10747, (1994). Briefly:

Prepare the DNA substrate to be subjected to the DNA shuffling reaction.Preparation may be in the form of simply purifying the DNA fromcontaminating cellular material, chemicals, buffers, oligonucleotideprimers, deoxynucleotides, RNAs, etc., and may entail the use of DNApurification kits as those provided by Qiagen, Inc., or by the Promega,Corp., for example.

Once the DNA substrate has been purified, it would be subjected to DnaseI digestion. About 2-4 ug of the DNA substrate(s) would be digested with0.0015 units of Dnase I (Sigma) per ul in 100 ul of 50 mM Tris-HCL, pH7.4/1 mM MgCl2 for 10-20 min. at room temperature. The resultingfragments of 10-50 bp could then be purified by running them through a2% low-melting point agarose gel by electrophoresis onto DE81ion-exchange paper (Whatmann) or could be purified using Microconconcentrators (Amicon) of the appropriate molecular weight cutoff, orcould use oligonucleotide purification columns (Qiagen), in addition toother methods known in the art. If using DE81 ion-exchange paper, the10-50 bp fragments could be eluted from said paper using 1M NaCl,followed by ethanol precipitation.

The resulting purified fragments would then be subjected to a PCRassembly reaction by re-suspension in a PCR mixture containing: 2 mM ofeach dNTP, 2.2 mM MgCl2, 50 mM KCl, 10 mM Tris●HCL, pH 9.0, and 0.1%Triton X-100, at a final fragment concentration of 10-30 ng/ul. Noprimers are added at this point. Taq DNA polymerase (Promega) would beused at 2.5 units per 100 ul of reaction mixture. A PCR program of 94 Cfor 60 s; 94 C for 30 s, 50-55 C for 30 s, and 72 C for 30 s using 30-45cycles, followed by 72 C for 5 min using an MJ Research (Cambridge,Mass.) PTC-150 thermocycler. After the assembly reaction is completed, a1:40 dilution of the resulting primerless product would then beintroduced into a PCR mixture (using the same buffer mixture used forthe assembly reaction) containing 0.8 um of each primer and subjectingthis mixture to 15 cycles of PCR (using 94 C for 30 s, 50 C for 30 s,and 72 C for 30 s). The referred primers would be primers correspondingto the nucleic acid sequences of the polynucleotide(s) utilized in theshuffling reaction. Said primers could consist of modified nucleic acidbase pairs using methods known in the art and referred to else whereherein, or could contain additional sequences (i.e., for addingrestriction sites, mutating specific base-pairs, etc.).

The resulting shuffled, assembled, and amplified product can be purifiedusing methods well known in the art (e.g., Qiagen PCR purification kits)and then subsequently cloned using appropriate restriction enzymes.

Although a number of variations of DNA shuffling have been published todate, such variations would be obvious to the skilled artisan and areencompassed by the invention. The DNA shuffling method can also betailored to the desired level of mutagenesis using the methods describedby Zhao, et al. (Nucl Acid Res., 25(6):1307-1308, (1997).

As described above, once the randomized pool has been created, it canthen be subjected to a specific screen to identify the variantpossessing the desired characteristic(s). Once the variant has beenidentified, DNA corresponding to the variant could then be used as theDNA substrate for initiating another round of DNA shuffling. This cycleof shuffling, selecting the optimized variant of interest, and thenre-shuffling, can be repeated until the ultimate variant is obtained.Examples of model screens applied to identify variants created using DNAshuffling technology may be found in the following publications: J. C.,Moore, et al., J. Mol. Biol., 272:336-347, (1997), F. R., Cross, et al.,Mol. Cell. Biol., 18:2923-2931, (1998), and A. Crameri., et al., Nat.Biotech., 15:436-438, (1997).

DNA shuffling has several advantages. First, it makes use of beneficialmutations. When combined with screening, DNA shuffling allows thediscovery of the best mutational combinations and does not assume thatthe best combination contains all the mutations in a population.Secondly, recombination occurs simultaneously with point mutagenesis. Aneffect of forcing DNA polymerase to synthesize full-length genes fromthe small fragment DNA pool is a background mutagenesis rate. Incombination with a stringent selection method, enzymatic activity hasbeen evolved up to 16000 fold increase over the wild-type form of theenzyme. In essence, the background mutagenesis yielded the geneticvariability on which recombination acted to enhance the activity.

A third feature of recombination is that it can be used to removedeleterious mutations. As discussed above, during the process of therandomization, for every one beneficial mutation, there may be at leastone or more neutral or inhibitory mutations. Such mutations can beremoved by including in the assembly reaction an excess of the wild-typerandom-size fragments, in addition to the random-size fragments of theselected mutant from the previous selection. During the next selection,some of the most active variants of thepolynucleotide/polypeptide/enzyme, should have lost the inhibitorymutations.

Finally, recombination enables parallel processing. This represents asignificant advantage since there are likely multiple characteristicsthat would make a protein more desirable (e.g. solubility, activity,etc.). Since it is increasingly difficult to screen for more than onedesirable trait at a time, other methods of molecular evolution tend tobe inhibitory. However, using recombination, it would be possible tocombine the randomized fragments of the best representative variants forthe various traits, and then select for multiple properties at once.

DNA shuffling can also be applied to the polynucleotides andpolypeptides of the present invention to decrease their immunogenicityin a specified host. For example, a particular variant of the presentinvention may be created and isolated using DNA shuffling technology.Such a variant may have all of the desired characteristics, though maybe highly immunogenic in a host due to its novel intrinsic structure.Specifically, the desired characteristic may cause the polypeptide tohave a non-native structure which could no longer be recognized as a“self” molecule, but rather as a “foreign”and thus activate a hostimmune response directed against the novel variant. Such a limitationcan be overcome, for example, by including a copy of the gene sequencefor a xenobiotic ortholog of the native protein in with the genesequence of the novel variant gene in one or more cycles of DNAshuffling. The molar ratio of the ortholog and novel variant DNAs couldbe varied accordingly. Ideally, the resulting hybrid variant identifiedwould contain at least some of the coding sequence which enabled thexenobiotic protein to evade the host immune system, and additionally,the coding sequence of the original novel variant that provided thedesired characteristics.

Likewise, the invention encompasses the application of DNA shufflingtechnology to the evolution of polynucleotides and polypeptides of theinvention, wherein one or more cycles of DNA shuffling include, inaddition to the gene template DNA, oligonucleotides coding for knownallelic sequences, optimized codon sequences, known variant sequences,known polynucleotide polymorphism sequences, known ortholog sequences,known homologue sequences, additional homologous sequences, additionalnon-homologous sequences, sequences from another species, and any numberand combination of the above.

In addition to the described methods above, there are a number ofrelated methods that may also be applicable, or desirable in certaincases. Representative among these are the methods discussed in PCTapplications WO 98/31700, and WO 98/32845, which are hereby incorporatedby reference. Furthermore, related methods can also be applied to thepolynucleotide sequences of the present invention in order to evolveinvention for creating ideal variants for use in gene therapy, proteinengineering, evolution of whole cells containing the variant, or in theevolution of entire enzyme pathways containing polynucleotides of theinvention as described in PCT applications WO 98/13485, WO 98/13487, WO98/27230, WO 98/31837, and Crameri, A., et al., Nat. Biotech.,15:436-438, (1997), respectively.

Additional methods of applying “DNA Shuffling” technology to thepolynucleotides and polypeptides of the present invention, includingtheir proposed applications, may be found in U.S. Pat. No. 5,605,793;PCT Application No. WO 95/22625; PCT Application No. WO 97/20078; PCTApplication No. WO 97/35966; and PCT Application No. WO 98/42832; PCTApplication No. WO 00/09727 specifically provides methods for applyingDNA shuffling to the identification of herbicide selective crops whichcould be applied to the polynucleotides and polypeptides of the presentinvention; additionally, PCT Application No. WO 00/12680 providesmethods and compositions for generating, modifying, adapting, andoptimizing polynucleotide sequences that confer detectable phenotypicproperties on plant species; each of the above are hereby incorporatedin their entirety herein for all purposes.

It will be clear that the invention may be practiced otherwise than asparticularly described in the foregoing description and examples.Numerous modifications and variations of the present invention arepossible in light of the above teachings and, therefore, are within thescope of the appended claims.

The entire disclosure of each document cited (including patents, patentapplications, journal articles, abstracts, laboratory manuals, books, orother disclosures) in the Background of the Invention, DetailedDescription, and Examples is hereby incorporated herein by reference.Further, the hard copy of the sequence listing submitted herewith andthe corresponding computer readable form are both incorporated herein byreference in their entireties.

1-20. (canceled)
 21. A method of diagnosing Parkinson's diseasecomprising: a) measuring the expression level of RNA encoding apolypeptide comprising the sequence of SEQ ID NO:2 or a fragment thereofin a Parkinson's substantia nigra test sample; and b) comparing saidexpression level of said RNA from a normal Parkinson's substantia nigrasample; wherein an increased expression level of said RNA in said testsample relative to the expression level of said RNA in said normalsample is indicative of Parkinson's disease or a predisposition toParkinson's disease.
 22. The method according to claim 21, wherein saidRNA measurement comprises specific hybridization between said RNA to amember of the group consisting of: a.) an isolated nucleic acidconsisting of SEQ ID NO:1; b.) an isolated nucleic acid consisting ofnucleotides 101 to 1873 of SEQ ID NO:1; c.) an isolated nucleic acidconsisting of nucleotides 104 to 1873 of SEQ ID NO:1; d.) an isolatednucleic acid consisting of nucleotides 104 to 1858 of SEQ ID NO:1; e.)an isolated nucleic acid consisting of nucleotides 302 to 1858 of SEQ IDNO:1; f.) isolated polynucleotide consisting of cDNA clone, HLLRCAR-1,contained in ATCC Deposit No: PTA-3434; g.) an isolated polynucleotideconsisting of at least 1104 contiguous nucleotides of SEQ ID NO:1,wherein the complementary sequence of said polynucleotide sequencehybridizes under stringent conditions to a polynucleotide comprisingnucleotides 104 to 1873 of SEQ ID NO:1, wherein said stringentconditions are at least as stringent as an overnight incubation at 42degree C. in a solution comprising 50% formamide, 5×SSC (750 mM NaCl, 75mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5× Denhardt'ssolution, 10% dextran sulfate, and 20 μg/ml denatured, sheared salmonsperm DNA, followed by washing the filters in 0.1×SSC at about 65 degreeC.; and h.) an isolated nucleic acid consisting of the complementarysequence of (a), (b), (c), (d), (e), (f), and (g).
 23. The methodaccording to claim 21, wherein said RNA measurement comprises specifichybridization between said RNA to an isolated nucleic acid consisting ofa complete complement of at least 19 contiguous nucleotides of SEQ IDNO:1, wherein said hybridization is performed under conditions at leastas stringent as hybridization in 2.2 mM MgCl2, 50 mM KCl, 10 mMTris●HCL, pH 9, and 0.1% Triton X-100 at 42 C.
 24. The method accordingto claim 23, wherein said RNA measurement further comprises a secondisolated nucleic acid consisting of at least 19 contiguous nucleotidesof SEQ ID NO:1, wherein said isolated nucleic acid of claim 23 isdirected to the antisense strand, and said second isolated nucleic acidis directed to the sense strand, and wherein said hybridization isfollowed by at least one amplification step.